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G.  P.  PUTNAM'S  SONS,  NEW  YORK  AND  LONDON 


ZTbe  Science  Series 

EDITED    BY 

ff»rofc0gor  3.  flDclKeen  Cattell,  flD.H.,  tf>b.2>. 

AND 

ff.  E. 


BACTERIA 


BACTERIA 

ESPECIALLY  AS  THEY  ARE    RELATED 
TO   THE   ECONOMY  OF   NATURE 

TO  INDUSTRIAL  PROCESSES 
AND  TO  THE   PUBLIC   HEALTH 


BY 


GEORGE    NEWMAN 

M.D.,  F.R.S.  (EDiN.),  D.V.H.  (CAMB.),  ETC. 

DEMONSTRATOR   OF   BACTERIOLOGY   IN    KING'S   COLLEGE,    LONDON 


ILLUSTRATED 


NEW  YORK 

G.  P.  PUTNAM'S   SONS 

LONDON 

JOHN  MURRAY 

1899 


IV 


GENERAL 


COPYRIGHT,  1899 

BY 

G.  P.  PUTNAM'S  SONS 


TTbc  fmfcfeevbocfcer  press,  IRew  H?orfe 


PREFACE 

THE  present  volume  is  not  a  record  of  original  work,  nor 
is  it  a  text-book  for  the  laboratory.  Theoretical  and 
practical  text-books  of  Bacteriology  plentifully  exist  both  in 
England  and  America.  There  are  two  large  works  widely 
used,  one  by  Professor  Crookshank,  entitled  Bacteriology  and 
Infective  Diseases,  the  other  by  Dr.  Sternberg,  A  Manual  of 
Bacteriology.  There  are  also,  in  English,  a  number  of  smaller 
works  by  Abbott,  Ball,  Hewlett,  Klein,  Macfarland,  Muir 
and  Ritchie,  and  Sims  Woodhead.  This  book  is  of  a  less 
technical  nature.  It  is  an  attempt,  in  response  to  the  editor 
of  the  series,  to  set  forth  a  popular  scientific  statement  of  our 
present  knowledge  of  bacteria.  /^Popular  science  is  a  some- 
what dangerous  quantity  with  which  to  deal.  On  the  one 
hand  it  may  become  too  popular,  on  the  other  too  technical. 
It  is  difficult  to  escape  the  Scylla  and  Charybdis  in  such 
a  voyage. 

I  am  much  indebted  to  Professor  Crookshank,  who,  in 
reading  the  manuscript,  has  helped  me  by  many  valuable 
criticisms.  My  thanks  are  also  due  to  Sir  C.  T.  D.  Acland, 
Bart.,  for  many  kind  suggestions,  and  to  Mr.  E.  J.  Spitta, 
M.R.C.S.,  who  has  been  good  enough  to  take  a  number  of 
excellent  photo-micrographs  forme.  Some  other  illustrations 
have  been  derived  from  the  Atlas  of  Bacteriology,  brought  out 
jointly  by  Messrs.  Slater  and  Spitta.  For  these  also  I  am 
glad  to  have  an  opportunity  of  expressing  my  thanks.  It 
should  be  understood  that  the  outline  drawings  are  only  of 
a  diagrammatic  nature. 

GEORGE  NEWMAN. 

LONDON,  1899. 


CONTENTS 


PAGE 

INDRODUCTION ix 

CHAPTER  I 
THE  BIOLOGY  OF  BACTERIA  i 


CHAPTER  II 
BACTERIA  IN  WATER     ..........       37 

CHAPTER  III 
BACTERIA  IN  THE  AIR  .         .        . 96 

CHAPTER  IV 
BACTERIA  AND  FERMENTATION in 

CHAPTER  V 
BACTERIA  IN  THE  SOIL 137 

CHAPTER  VI 
BACTERIA  IN  MILK,  MILK  PRODUCTS,  AND  OTHER  FOODS          .        .     178 

CHAPTER  VII 
THE  QUESTION  OF  IMMUNITY  AND  ANTITOXINS 240 

CHAPTER  VIII 
BACTERIA  AND  DISEASE 264 

CHAPTER  IX 
DISINFECTION         ...........     322 

APPENDIX  337 


ILLUSTRATIONS 


[Illustrations  starred  (*)  are  reproduced  by  permission  of  the  Scientific  Press 
from  Drs.  Spitta  and  Slater's  Atlas  of  Bacteriology.] 

PAGE 

VARIOUS  FORMS  OF  BACTERIA 9 

SARCINA 10 

NORMAL  AND  PLEOMORPHIC  FORMS  OF  TUBERCLE        ....  13 

BACILLI,  SHOWING  FLAGELLA 15 

VARIOUS  FORMS  OF  SPORE  FORMATION  AND  FLAGELLA        .         .         .18 

POTATO  IN  A  Roux  TUBE  PREPARED  FOR  CULTIVATION     ...  22 

STAPHYLOCOCCUS  PYOGENES  AUREUS  INCUBATOR          .        .       to  face  22 

CULTURE  MEDIA  READY  FOR  INOCULATION 23 

INOCULATING  NEEDLES 24 

PASTEUR'S  LARGE  INCUBATOR  FOR  CULTIVATION  AT  ROOM  TEMPER- 
ATURE       to  face  24 

METHOD  OF  PRODUCING  HYDROGEN  BY  KIPP'S  APPARATUS  FOR  CUL- 
TIVATION OF  ANAEROBES 27 

ANAEROBIC  CULTURE     .         .        . 28 

KOCH'S  STEAM  STERILISER 31 

LEVELLING  APPARATUS  FOR  KOCH'S  PLATE 40 

MOIST  CHAMBER  IN  WHICH  KOCH'S  PLATES  ARE  INCUBATED       .         .  41 

HOT-AIR  STERILISER      ..........  42 

THE  HANGING  DROP 44 

DRYING  STAGE  FOR  FIXING  FILMS 45 

TYPES  OF  LIQUEFACTION  OF  GELATINE         ......  47 

WOLFHOGEL'S  COUNTER 49 

PETRI'S  DISH 50 

BERKEFELD  FILTER 52 

APPARATUS   FOR   FILTERING   WATER   TO    FACILITATE   ITS    BACTERIO- 
LOGICAL EXAMINATION to  face  52 

BACTERIA  OF  TYPHOID  FEVER 56 

BACILLUS  COLI  COMMUNIS 60 

THE  COMMA-SHAPED  BACILLUS  OF  CHOLERA 66 

*BACILLUS  TYPHOSUS to  face  66 

*BACILLUS  TYPHOSUS                               66 


V  i  i  i  ILL  US  TRA  TIONS 


*BACILLUS  COLI  COMMUNIS  .......  66 

*BACILLUS  MYCOIDES 66 

PASTEUR-CHAMBERLAND  FILTER So 

PROTEUS  VULGARIS         ..........  86 

BACILLUS  ENTERIDITIS  SPOROGENES  .......  86 

A  PLAN  OF  SEPTIC  TANK  AND  FILTER-BEDS 91 

FILTER-BEDS 94 

MIQUEL'S  FLASK 97 

SEDGWICK'S  SUGAR-TUBE 99 

SEDGWICK'S  TUBE  .  .  .  .  .  .  .  .  .  .  100 

SACCHAROMYCES  CEREVISLE 117 

ASCOSPORE  FORMATION 120 

GYPSUM  BLOCK 121 

YEAST to  face  122 

ASCOSPORE  FORMATION  IN  YEAST 122 

NITROGEN-FIXING  BACTERIA  FROM  ROOTLET  NODULES  .  "  122 

*BACILLUS  OF  TETANUS "122 

SACCHAROMYCES  ELLIPSOIDEUS 126 

SACCHAROMYCES  PASTORIANUS 126 

BACILLUS  ACIDI  LACTICI 131 

BACCILLUS  BUTYRICUS 133 

KIPP'S  APPARATUS          ..........  140 

FRANKEL'S  TUBE  ............  141 

BUCHNER'S  TUBE 141 

A  METHOD  OF  GROWING  CULTIVATIONS  IN  A  VACUUM  OVER  PYROGALLIC 

SOLUTION 143 

MlCROCOCCUS  FROM  SOIL 151 

NITROUS  ORGANISM to  face  158 

NITRIC  ORGANISM  .        .        .        .        .        .        .        .        .            "  158 

NITROGEN-FIXING  ORGANISM  FROM  SECRETION  OF  ROOT-NODULES,   "  158 

ROOTLET  OF  PEA  WITH  NODULES         .......  163 

NlTROGEN-FlXING  BACTERIA  IN  SlTU  IN  NODULE  ON  ROOTLET  OF 

A  PEA to  face  164 

NlTROGEN-FlXING      BACTERIA     IN     SlTU      IN     ROOTLET-NODULE      OF     A 

PEA.         ..........         to  face  164 

NlTROGEN-FlXING  BACTERIA  IN  SlTU  IN  ROOT-NODULE  OF  A  PEA,            "  164 

BACILLUS  OF  TETANUS 170 

BACILLUS  OF  SYMPTOMATIC  ANTHRAX  .         .         .        .        .        .172 

BACILLUS  OF  MALIGNANT  OEDEMA         .        .         .        .        .         .         .172 

A  CENTRIFUGE 228 

SUSPENDED  SPINAL  CORD 255 

FLASK  USED  IN  THE  PREPARATION  OF  THE  TOXIN  OF  DIPHTHERIA  .  262 

*BACILLUS  TUBERCULOSIS to  face  280 


ILL  US  TRA  TIONS  \  x 

PAGE 

*BACILLUS  TUBERCULOSIS      ...                         .                    "  280 

*STREPTOCOCCUS  PYOGENES  .......            "  280 

*  BACILLUS  ANTHRACIS   .        .        .        ..        .        .        .        .            "  280 

FLASK  USED  IN  THE  PREPARATION  OF  TUBERCULIN     ....  282 

BACILLUS  OF  DIPHTHERIA 289 

TYPES  OF  STREPTOCOCCUS 298 

MICROCOCCUS  TETRAGONUS 299 

DIPLOCOCCUS  OF  NEISSER 300 

BACILLUS  OF  ANTHRAX  AND  BLOOD  CORPUSCLES         ....  302 

THREADS  OF  BACILLUS  ANTHRACIS,  SHOWING  SPORES         .        .        .  302 

BACILLUS  OF  PLAGUE     ..........  306 

*BACILLUS  OF  PLAGUE   ........        to  face  310 

*BACILLUS  OF  LEPROSY "  310 

STREPTOTHRIX  ACTINOMYCES         .        .        .        .        .        .            "  310 

BACILLUS  MALLEI "  310 

DIPLOCOCCUS  OF  PNEUMONIA 312 

BACILLUS  OF  INFLUENZA 315 


INTRODUCTION 

WE  live  in  a  world  that  is  teeming  with  life.  From  the 
earliest  times  of  man  that  life  has  been  studied  and 
the  observations  recorded.  Thus  there  has  slowly  come  to 
be  a  considerable  accumulation  of  knowledge  concerning  the 
various  forms  (morphology)  and  functions  (physiology)  of 
organised  life.  This  we  call  the  science  of  biology.  It  has 
for  its  object  the  study  of  organic  beings,  and  for  its  end  the 
knowledge  of  the  laws  of  their  organisation  and  activity. 
Slowly,  too,  in  the  midst  of  this  gradual  accumulation  of 
facts,  we  begin  to  see  incoherence  becoming  coherent,  chaos 
becoming  cosmos,  chance  and  accident  becoming  law. 
Further,  the  contemplation  and  comprehension  which  built 
up  the  edifice  of  modern  biology  is  assuming  a  new  relation- 
ship to  practical  life.  Biology  can  no  longer  be  considered 
only  as  an  academic  occupation  or  as  a  theoretical  pabulum 
upon  which  the  leisured  mind  may  ruminate.  With  rapid 
strides  and  determined  face  this  giant  of  knowledge  has 
marched  into  the  arena  of  practical  politics.  The  world  is 
opening  its  eyes  to  a  reality  which  it  had  mistaken  for  a  vision. 
This  application  of  biology  to  life  and  its  problems  has  in 
recent  years  been  nowhere  more  marked  than  in  the  realm 
of  bacteriology.  This  comparatively  new  science,  associated 
with  the  great  names  of  Pasteur,  Koch,  and  Lister,  furnishes 
hideed  a  stock  illustration  of  the  applicability  of  pure  biology. 
''Turn  where  we  will,  we  shall  find  the  work  of  the  unseen 
hosts  of  bacteria  daily  claiming  more  and  more  attention 
from  practical  people.  Thus  biology,  even  when  clothed  in 
the  form  of  microscopic  cells,  is  coming  to  occupy  a  new 
place  in  the  minds  of  men.  "  Its  evolution,"  as  Professor 


xi  i  IN  TROD  UCTION 

Patrick  Geddes  declares,  "  forms  part  of  the  general  social 
evolution."  Certainly  its  recent  rapid  development  forms  a 
remarkable  feature  in  the  practical  science  of  bur  time.  Not 
only  in  the  diagnosis  and  treatment  of  disease,  nor  even  in 
the  various  applications  of  preventive  medicine,  but  in  ever- 
increasing  degree  and  sphere,  micro-organisms  are  recognised 
as  agents  of  utility  or  otherwise  no  longer  to  be  ignored. 
They  occur  in  our  drinking  water,  in  our  milk  supply,  in  the 
air  we  breathe.  They  ripen  cream,  and  flavour  butter.  They 
purify  sewage,  and  remove  waste  organic  products  from  the 
land.  They  are  the  active  agents  in  a  dozen  industrial 
fermentations.  They  assist  in  the  fixation  of  free  nitrogen, 
and  they  build  up  assimilable  compounds.  Their  activity 
assumes  innumerable  phases  and  occupies  many  spheres, 
more  frequently  proving  themselves  beneficial  than  injurious. 
They  are  both  economic  and  industrious  in  the  best  biological 
sense  of  the  terms. 

Yet  bacteriology  has  its  limitations.  It  is  well  to  recognise 
this,  for  the  new  science  has  in  some  measure  suffered  in  the 
past  from  over-zealous  friends.  It  cannot  achieve  everything 
demanded  of  it,  nor  can  it  furnish  a  cause  for  every  disease. 
It  is  a  science  fuller  of  hope  than  proved  and  tested  know- 
ledge. We  are  as  yet  only  upon  the  threshold  of  the  matter. 
As  in  the  neighbouring  realm  of  chemistry,  it  is  to  be  feared 
that  bacteriology  has  not  been  without  its  alchemy.  The 
interpretations  and  conclusions  which  have  been  drawn  from 
time  to  time  respecting  bacteriological  work  have  led  to 
alarmist  views  which  have  not,  by  later  investigation,  been 
fully  supported.  Again,  the  science  has  had  devotees  who 
have  fondly  believed,  like  the  alchemists,  that  the  twin  secret 
of  transmuting  the  baser  metals  into  gold  and  of  indefinitely 
prolonging  human  life  was  at  last  to  be  known.  But  neither 
the  worst  fears  of  the  alarmist  nor  the  most  sanguine  hopes 
of  the  alchemist  have  been  verified.  Science,  fortunately, 
does  not  progress  at  such  speed,  or  with  such  kindly  accom- 


INTRODUCTION  xiii 

modation.  It  holds  many  things  in  its  hands,  but  not  finally 
life  or  death.  It  has  not  yet  brought  to  light  either  "the 
philosopher's  stone"  or  "  the  vital  essence." 

What  has  already  been  said  affords  ample  reason  for  a 
wider  dissemination  of  the  elementary  facts  of  bacteriological 
science.  But  there  are  other  reasons  of  a  more  practical 
nature.  Municipalities  are  expending'  public  moneys  in 
water  analysis,  in  the  examination  of  milk,  in  the  inspection 
of  cows  and  dairies,  in  the  bacterial  treatment  of  sewage,  and 
in  disinfection  and  other  branches  of  public  health  adminis- 
tration. Again,  the  newly  formed  National  Association 
for  the  Prevention  of  Tuberculosis,  our  increasing  colonial 
possessions  with  their  tropical  diseases,  even  medical  science 
itself,  which  is  year  by  year  becoming  more  preventive,  make 
an  increasing  claim  upon  public  opinion.  The  successful 
accomplishment  and  solution  of  these  questions  depend  in 
a  measure  upon  an  educated  public  opinion  respecting  the 
elements  of  bacteriology.  Recently  it  was  urged  that  "the 
first  elements  of  bacteriology  should  be  shadowed  forth  in 
the  primary  school."  This  course  was  advised  owing  to  such 
knowledge  being  of  value  to  those  engaged  in  dairying.  As 
we  shall  point  out  at  a  later  stage,  many  of  the  undesirable 
changes  occurring  in  milk  are  due  to  bacteria,  even  as  the 
success  of  the  butter  and  cheese  industries  depends  on  the 
use  and  control  of  the  fermentative  processes  due  to  their 
action.  Much  of  the  uncertainty  attending  the  manufacture 
of  dairy  products  can  only  be  abolished  by  the  careful 
application  of  some  knowledge  of  the  flora  of  milk.  In 
Denmark  and  in  Scandinavia  the  importance  of  such 
knowledge  is  realised  and  acted  upon.  America,  too,  has 
not  been  slow  to  respond  to  these  needs  ;  but  in  England 
comparatively  little  has  been  done  in  this  direction.2 

1  The  Contemporary  Review,  November,  1897,  p.  719. 

2  Some  notable  exceptions  are  found  in  the  work  of  the  Bath  and  West  of 
England  Society,  Lord  Vernon's  model  dairy,  and  the  Essex  County  Council 
Bacteriological  Teaching  Laboratory. 


XIV  INTRODUCTION 

Whilst  there  can  be  no  doubt  as  to  the  advantage  of  a 
wider  dissemination  of  the  ascertained  facts  concerning 
bacteria,  it  should  be  borne  in  mind  that  only  patient,  skilled 
observation  and  experimental  research  in  well-equipped 
laboratories  can  advance  this  branch  of  science,  or  indeed 
train  bacteriologists.  The  lives  of  Darwin  and  of  Pasteur 
adequately  illustrate  this  truth.  Yet  it  is  observable  that 
States  and  public  bodies  are  slow  to  act  upon  it,  and  fre- 
quently in  the  past  the  most  useful  and  substantial  support 
for  the  advancement  of  science  has  been  forthcoming  only 
from  private  sources.  As  the  world  learns  its  intimate 
relation  to  science  and  the  interdependence  between  its  life 
and  scientific  truth,  it  may  be  expected  more  heartily  to 
support  science. 


BACTERIA 


BACTERIA 


CHAPTER  I 

THE  BIOLOGY  OF  BACTERIA  ' 

THE  first  scientist  who  demonstrated  the  existence  of 
micro-organisms  was  Antony  von  Leeuwenhoek.  He 
was  born  at  Delft,  in  Holland,  in  1632,  and  enthusiastically 
pursued  microscopy  with  primitive  instruments.  He  corro- 
borated Harvey's  discovery  of  the  circulation  of  the  blood  in 
the  web  of  a  frog's  foot ;  he  defined  the  red  blood  corpuscles 
of  vertebrates,  the  fibres  of  the  lens  of  the  human  eye,  the 
scales  of  the  skin,  and  the  structure  of  hair.  He  was  neither 
educated  nor  trained  in  science,  but  in  the  leisure  time  of  his 
occupation  as  a  linen-draper  he  learned  the  art  of  grinding 
lenses,  in  which  he  became  so  proficient  that  he  was  able  to 
construct  a  microscope  of  greater  power  than  had  been 
previously  manufactured.  The  compound  microscope  dates 
from  1 590,  and  when  Leeuwenhoek  was  about  forty  years  old, 
Holland  had  already  given  to  the  world  both  microscope 
and  telescope.  Robert  Hooke  did  for  England  what  Hans 
Janssen  had  done  for  Holland,  and  established  the  same 

1  We  propose  throughout  to  use  the  term  bacterium  (pi.  bacteria)  in  its  generic 
meaning,  unless  especially  stated  to  the  contrary.  It  will  also  be  synonymous 
with  the  terms  microbe,  germ,  and  micro-organism.  The  term  bacillus  will,  of 
course,  be  restricted  to  a  rod-shaped  bacterium. 


2  BACTERIA 

conclusion  that  Leeuwenhoek  arrived  at  independently,  viz., 
that  a  simple  globule  of  glass  mounted  between  two  metal 
plates  and  pierced  with  a  minute  aperture  to  allow  rays  of 
light  to  pass  was  a  contrivance  which  would  magnify  more 
highly  than  the  recognised  microscopes  of  that  day.  It  was 
with  some  such  instrument  as  this  that  the  first  micro- 
organisms were  observed  in  a  drop  of  water.  It  was  not 
until  more  than  a  hundred  years  later  that  these  "  animal- 
cules," as  they  were  termed,  were  thought  to  be  anything 
more  than  accidental  to  any  fluid  or  substance  containing 
them.  Plenciz,  of  Vienna,  was  one  of  the  first  to  conceive 
the  idea  that  decomposition  could  only  take  place  in  the 
presence  of  some  of  these  "  animalcules."  This  was  in  the 
middle  of  the  eighteenth  century.  Just  about  a  century  later, 
by  a  series  of  important  discoveries,  it  was  established  beyond 
dispute  that  these  micro-organisms  had  an  intimate  causal 
relation  to  fermentation,  putrefaction,  and  infectious  diseases. 
Spallanzani,  Pasteur,  and  Tyndall  are  the  three  who  more 
than  others  contributed  to  this  discovery.  Spallanzani  was 
an  Italian,  who  studied  at  Bologna,  and  was  in  1754 
appointed  to  the  chair  of  logic  at  Reggio.  But  his  inclin- 
ations led  him  into  the  realm  of  natural  history.  Amongst 
other  things,  his  attention  was  directed  to  the  doctrine  of 
spontaneous  generation,  which  had  been  propounded  by 
Needham  a  few  years  previously.  In  1768  Spallanzani 
became  Professor  of  Natural  History  at  Pavia,  and  whilst 
there  he  demonstrated  that  if  infusions  of  vegetable  matter 
were  placed  in  flasks  and  hermetically  sealed,  and  then 
brought  to  the  boiling  point,  no  living  organisms  could 
thereafter  be  detected,  nor  did  the  vegetable  matter  decom- 
pose. When,  however,  the  flasks  were  very  slightly  cracked, 
and  air  gained  admittance,  then  invariably  both  organisms 
and  decomposition  appeared.  Schwann,  the  founder  of  the 
cell-theory,  and  Schulze,  both  showed  that  if  the  air  gaining 
access  to  the  flask  were  either  passed  through  highly  heated 


THE  BIOLOGY  OF  BACTERIA  3 

tubes  or  drawn  through  strong  acid  the  result  was  the  same 
as  if  no  air  entered  at  all,  viz.,  no  organisms  and  no  decom- 
position. The  result  of  these  investigations  was  that  scien- 
tific men  began  to  believe  that  no  form  of  life  arose  de  novo 
(abiogenesis),  but  had  its  source  in  previous  life  (biogenesis}. 
It  remained  to  Pasteur  and  Tyndall  to  demonstrate  this 
beyond  dispute,  and  to  put  to  rout  the  fresh  arguments  for 
spontaneous  generation  which  Pouchet  had  advanced  as  late 
as  1859.  Pasteur  collected  the  floating  dust  of  the  air,  and 
found  by  means  of  the  microscope  many  organised  particles, 
which  he  sowed  on  suitable  infusions,  and  thus  obtained  rich 
crops  of  "  animalculae."  He  also  demonstrated  that  these 
organisms  existed  in  different  degrees  in  different  atmo- 
spheres, few  in  the  pure  air  of  the  Mer  de  Glace,  more  in 
the  air  of  the  plains,  most  in  the  air  of  towns.  He  further 
proved  that  it  was  not  necessary  to  insist  upon  hermetic 
sealing  or  cotton  filters  to  keep  these  living  organisms  in  the 
air  from  gaining  access  to  a  flask  of  infusion.  If  the  neck  of 
the  flask  were  drawn  out  into  a  long  tube  and  turned  down- 
wards, and  then  a  little  upwards,  even  though  the  end  be  left 
open,  no  contamination  gained  access.  Hence,  if  the  infusion 
were  boiled,  no  putrefaction  would  occur.  The  organisms 
which  fell  into  the  open  end  of  the  tube  were  arrested  in  the 
condensation  water  in  the  angle  of  the  tube ;  but  even  if  that 
were  not  so,  the  force  of  gravity  acting  upon  them  prevented 
them  from  passing  up  the  long  arm  of  the  tube  into  the  neck 
of  the  flask.  A  few  years  after  Pasteur's  first  work  on  this 
subject  Tyndall  conceived  a  precise  method  of  determining 
the  absence  or  presence  of  dust  particles  in  the  air  by  passing 
a  beam  of  sunlight  through  a  glass  box  before  and  after  its 
walls  had  been  coated  with  glycerine.  Into  the  floor  of  the 
box  were  fixed  the  mouths  of  flasks  of  infusion.  These 
were  boiled,  after  which  they  were  allowed  to  cool,  and 
might  then  be  kept  for  weeks  or  months  without  putrefying 
or  revealing  the  presence  of  germ  life.  Here  all  the  con- 


4  BACTERIA 

ditions  of  the  infusions  were  natural,  except  that  in  the  air 
above  them  there  was  no  dust. 

The  sum-total  of  result  arising  from  all  these  investigations 
was  to  the  effect  that  no  spontaneous  generation  was  possible, 
that  the  atmosphere  contained  unseen  germs  of  life,  that  the 
smallest  of  organisms  responded  to  the  law  of  gravitation  and 
adhered  to  moist  surfaces,  and  that  micro-organisms  were  in 
some  way  or  other  the  cause  of  putrefaction. 

The  final  refutation  of  the  hypothesis  of  spontaneous 
generation  was  followed  by  an  awakened  interest  in  the 
unseen  world  of  micro-organic  life.  Investigations  into 
fermentation  and  putrefaction  followed  each  other  rapidly, 
and  in  1863  Davaine  claimed  that  Pollender's  bacillus  of 
anthrax,  which  was  found  in  the  blood  and  body  tissues  of 
animals  dead  of  anthrax,  was  the  cause  of  that  disease.  From 
that  time  to  this  in  every  department  of  biology  bacteria  have 
been  increasingly  found  to  play  an  important  part.  They 
cause  changes  in  milk,  and  flavour  butter ;  they  decompose 
animal  matter,  yet  build  up  the  broken-down  elements  into 
compounds  suitable  for  use  in  nature's  economy;  they  assist 
in  the  fixation  of  free  nitrogen ;  they  purify  sewage  ;  in 
certain  well-established  cases  they  are  the  cause  of  specific 
disease,  and  in  many  other  cases  they  are  the  likely  cause. 
No  doubt  the  disposal  of  spontaneous  generation  did  much 
to  arouse  interest  in  this  branch  of  science.  Yet  it  must 
not  be  forgotten  that  the  advance  of  the  microscope  and 
bacteriological  method  and  technique  have  played  a  large 
share  in  this  development.  The  sterilisation  of  culture  fluids 
by  heat,  the  use  of  aniline  dyes  as  staining  agents,  the  intro- 
duction of  solid  culture  media  (like  gelatine  and  agar),  and 
Koch's  "  plate  "  method  have  all  contributed  not  a  little  to 
the  enormous  strides  of  bacteriology.  Owing  to  its  relation 
to  disease,  physicians  have  entered  keenly  into  the  arena  of 
bacteriological  research.  Hence,  from  a  variety  of  causes,  it 
has  come  about  that  the  advance  has  been  phenomenal. 


THE  BIOLOGY  OF  BACTERIA  5 

We  shall  now  take  up  a  number  of  points  in  the  biology 
of  bacteria  which  call  for  early  attention,  and  which  are 
mostly  the  outcome  of  comparatively  recent  work  on  the 
subject. 

The  Place  of  Bacteria  in  Nature.  As  we  have  seen,  for  a 
considerable  period  of  time  after  their  first  detection  these 
unicellular  organisms  were  considered  to  be  members  of  the 
animal  kingdom.  As  late  as  1838,  when  Ehrenberg  and 
Dujardin  drew  up  their  classification,  bacteria  were  placed 
among  the  Infusorians.  This  was  in  part  due  to  the  powers 
of  motion  which  these  observers  detected  in  bacteria.  It 
is  now,  of  course,  recognised  that  animals  have  no  monopoly 
of  motion.  But  what,  after  all,  are  the  differences  between 
animals  and  vegetables  so  low  down  in  the  scale  of  life? 
Chiefly  two  :  there  is  a  difference  in  life-history  (in  structure 
and  development),  and  there  is  a  difference  in  diet.  A  plant 
secures  its  nourishment  from  much  simpler  elements  than  is 
the  case  with  animals ;  for  example,  it  obtains  its  carbon 
from  the  carbonic  acid  gas  in  air  and  water.  This  it  is 
able  to  do,  as  regards  the  carbon,  by  means  of  the  green 
colouring  matter  known  as  chlorophyll,  by  the  aid  of  which, 
with  sunlight,  carbonic  acid  is  decomposed  in  the  chlorophyll 
corpuscles,  the  oxygen  passing  back  into  the  atmosphere,  the 
carbon  being  stored  in  the  plant  in  the  form  of  starch  or 
other  organic  compound.  The  supply  of  carbon  in  the 
chlorophyll-free  plants,  among  which  are  the  bacteria,  is 
obtained  by  breaking  up  different  forms  of  carbohydrates. 
Besides  albumen  and  peptone,  they  use  sugar  and  similar 
carbohydrates  and  glycerine  as  a  source  of  carbon.  Many 
of  them  also  have  the  capacity  of  using  organic  matters  of 
complex  constitution  by  converting  such  into  water,  carbonic 
acid  gas,  and  ammonia.  Their  hydrogen  comes  from  water, 
their  nitrogen  from  the  soil,  chiefly  in  the  form  of  nitrates. 
From  the  soil,  too,  they  obtain  other  necessary  salts.  Now 
all  these  substances  are  in  an  elementary  condition,  and  as 


6  BACTERIA 

such  plants  can  absorb  them.  Animals,  on  the  other  hand, 
are  only  able  to  utilise  compound  food  products  which  have 
been,  so  to  speak,  prepared  for  them  ;  for  example,  albu- 
minoids and  proteids.  They  cannot  directly  feed  upon  the 
elementary  substances  forming  the  diet  of  vegetables.  This 
distinction,  however,  did  not  at  once  clear  up  the  difficult 
matter  of  the  classification  of  bacteria.  It  is  true,  they 
possess  motion,  are  free  from  chlorophyll,  and  even  feed 
occasionally  upon  products  of  decomposition — three  physio- 
logical characters  which  would  ally  them  to  the  animal 
kingdom.  Yet  by  their  structure  and  capsule  of  cellulose 
and  by  their  life-history  and  mode  of  growth  they  un- 
mistakably proclaim  themselves  to  be  of  the  vegetable 
kingdom.  In  1853  Cohn  arrived  at  a  conclusion  to  this 
effect,  and  since  that  date  they  have  become  more  and  more 
limited  in  classification  and  restricted  in  definition. 

Even  yet,  however,  we  are  far  from  a  scientific  classifi- 
cation for  bacteria.  Nor  is  this  matter  for  surprise.  The 
development  in  this  branch  of  biology  has  been  so  rapid 
that  it  has  been  impossible  to  assimilate  the  facts  collected. 
The  facts  themselves  by  their  remarkable  variety  have  not 
aided  classification.  Names  which  a  few  years  ago  were 
applied  to  individual  species,  like  Bacillus  subtilis,  or 
Bacterium  termo,  or  Bacillus  coli,  are  now  representative, 
not  of  individuals,  but  of  families  and  groups  of  species. 
Again,  isolated  characteristics  of  certain  microbes,  such  as 
motility,  power  of  liquefying  gelatine,  size,  colour,  and  so 
forth,  which  at  first  sight  might  appear  as  likely  to  form  a 
basis  for  classification,  are  found  to  vary  not  only  between 
similar  germs,  but  in  the  same  germ.  Different  physical 
conditions  have  so  powerful  an  influence  upon  these  micro- 
scopic cells  that  their  individual  characters  are  constantly 
undergoing  change.  For  example,  bacteria  in  old  cultures 
assume  a  different  size,  and  often  a  different  shape,  from 
younger  members  of  precisely  the  same  species ;  Bacillus 


THE  BIOLOGY  OF  BACTERIA  7 

pyocyaneus  produces  a  green  to  olive  colour  on  gelatine, 
but  a  brown  colour  on  potato  ;  the  bacillus  of  Tetanus  is 
virulently  pathogenic,  and  yet  may  not  act  thus  unless  in 
company  with  certain  other  micro-organisms.  Hence  it  will 
at  once  appear  to  the  student  of  bacteriology  that,  though 
there  is  great  need  for  classification  amongst  the  six  or 
seven  hundred  species  of  microbes,  our  present  knowledge 
of  their  life-history  is  not  yet  advanced  enough  to  form 
more  than  a  provisional  arrangement. 

We  know  that  bacteria  are  allied  to  moulds  on  the  one 
hand  and  yeasts  on  the  other,  and  that  they  have  no  dif- 
ferentiation into  root,  stem,  or  leaf ;  we  know  that  they  are 
fungi  (having  no  chlorophyll),  in  which  no  sexual  reproduc- 
tion occurs,  and  that  their  mode  of  multiplication  is  by 
division.  From  such  facts  as  these  we  may  build  up  a 
classification  as  follows  : — 

VEGETABLE  KINGDOM. 


Ill  I 

Thallophyta.         Muscineae.  Pteridophyta.        Phanerogamia. 

[=  The  lowest  forms 
of  vegetable  life.     No 
differentiation     into 
root,   stem,   or  leaf.] 

Protophyta. 
[=  No  sexual  reproduction.] 

I  | 

Algse.  Fungi. 

[=  Chlorophyll  [=  No  chlorophyll.] 
present.] 


I    I     I     I     I     I     I     I     I 


Schizomycetes 

[=  multiplication  by  cell 

division  or  by  spores] 


r(i)  Coccaceae  1 — round  cells. 

(2)  Bacteriaceae — rods  and  threads. 


Bacteria. 


or 

(3)  Leptotrichese. 


Cladotrichese.  ) 


[•  Higher  Bacteria. 


1  Migula  has  recently  (1896)  suggested  that  the  Schizomycetes  should  be  subdivided  into 
Coccaceee,  Bacteriacete^  Spirillacea  (spirilla,  spirochoeta),  Chlamydobacteriacefe  (Streptothrix, 
Crenothrix,  Cladothrix),  and  Beggiatoa. 


8  BACTERIA 

Structure  and  Form.  Having  now  located  micro-organisms 
in  the  economy  of  nature,  we  may  proceed  to  describe  their 
subdivisions  and  form.  For  practical  convenience  rather 
than  academic  accuracy,  we  may  accept  the  simple  division 
of  the  family  of  bacteria  into  three  chief  forms,  viz.: — 
(  (i)  Round  cell  form — coccus. 

Lower  Bacteria  •<  (2)  Rod  form — bacillus. 

(  (3)  Thread  form — spirillum. 

Higher  Bacteria — Leptothrix,  Streptothrix, Cladothrix, etc. 

A  classification  dependent  as  this  is  upon  the  form  alone 
is  not  by  any  means  ideal,  for  it  ignores  all  the  higher  and 
complicated  functions  of  bacteria,  but  it  is,  as  we  have  said, 
practically  convenient. 

I.  The  Coccus.  This  is  the  group  of  round  cells.  They 
vary  in  size  as  regards  species,  and  as  regards  the  conditions, 
artificial  or  natural,  under  which  they  have  been  grown. 
Some  are  less  than  2g^OTr  of  an  inch  in  diameter ;  others  are 
half  as  large  again,  if  the  word  large  may  be  used  to  describe 
such  minute  objects.  No  regular  standard  can  be  laid  down 
as  reliable  with  regard  to  their  size.  Hence  the  subdivisions 
of  the  cocci  are  dependent  not  upon  the  individual  elements 
so  much  as  upon  the  relation  of  those  elements  to  each  other. 
A  simple  round  cell  of  approximately  the  size  already  named 
is  termed  a  micrococcus  (tempos,  small).  Certain  species  of 
micrococci  always  or  almost  always  occur  in  pairs,  and  such 
a  combination  is  termed  a  diplococcus.  Some  diplococci  are 
united  by  a  thin  capsule,  which  may  be  made  apparent  by 
special  methods  of  staining;  of  others  no  limiting  or  uniting 
membrane  can  be  seen  with  the  ordinary  high  powers  of  the 
microscope.1  Again,  one  frequently  finds  a  species  which  is 
exactly  described  by  saying  that  two  micrococci  are  in  con- 
tact with  each  other,  and  move  and  act  as  one  individual, 
but  otherwise  show  no  alteration  ;  whilst  others  are  seen 

1  A  one-twelfth  oil  immersion  lens  is  requisite  for  the  study  of  the  lower 
bacteria. 


THE  BIOLOGY  OF  BACTERIA  9 

which  show  a  flattening  of  the  side  of  each  micrococcus 
which  is  in  relation  to  its  partner.  Perhaps  the  diplococci  in 
an  even  greater  degree  than  the  micrococci  respond  to  ex- 
ternal conditions  both  as  regards  size  and  shape.  It  must 
further  be  borne  in  mind  that  a  dividing  micrococcus  assumes 
the  exact  appearance  of  a  diplococcus  during  the  transition 
stage  of  the  fission.  Hence,  with  the  exception  of  several 
well-marked  species  of  diplococci,  this  form  is  somewhat 


9  9 

VARIOUS  FORMS  OF  BACTERIA 

i.  Micrococcus  2.  Diplococcus  3.  Streptococcus 

4.  Staphylococcus  5.  Leuconostoc,  showing  Arthrospores 

6.  Merismopedia  7.  Sarcina  8.  Bacilli 

9.  Spirillum 

arbitrary.  The  third  kind  of  micrococcus  is  that  formed  by 
a  number  of  elements  in  a  twisted  chain,  named  streptococcus 
(ffTpSTtros,  twisted).  This  form  is  produced  by  cells  dividing 
in  one  axis,  and  remaining  in  contact  with  each  other.  It 
occurs  in  a  number  of  different  species,  or  what  are  supposed 


10  BACTERIA 

by  many  authorities  to  be  different  species,  owing  to  their 
different  effects.  Morphologically  all  the  streptococci  are 
similar,  though  a  somewhat  abortive  attempt  was  once  made 
to  divide  them  into  two  groups,  according  to  whether  they 
were  long  chains  or  short.  As  a  matter  of  fact,  the  length 
of  streptococci  depends  in  some  cases  upon  biological  prop- 
erties, in  others  upon  external  treatment  or  the  medium  of 
cultivation  which  has  been  used.  Sometimes  they  occur  as 
straight  chains  of  only  half  a  dozen  elements ;  at  other  times 
they  may  contain  thirty  to  forty  elements,  and  twist  in 


SARCINA 

various  ways,  even  forming  rosaries.  The  elements,  too, 
differ  not  only  in  size,  but  in  shape,  appearing  occasionally 
as  oval  cells  united  to  each  other  at  their  sides.  The  fourth 
form  is  constituted  by  the  micrococci  being  arranged  in 
masses  like  grapes,  the  staphylococcus  (ar&q>i$k{s9  a  bunch  of 
grapes).  The  elements  are  often  smaller  than  in  the  strep- 
tococcus, and  the  name  itself  describes  the  arrangement. 
There  is  no  matrix  and  no  capsule.  This  is  the  commonest 
organism  found  in  abscesses,  etc.  The  sarcina  is  best 
classified  amongst  the  cocci,  for  it  is  composed  of  them,  in 
packets  of  four  or  multiples  of  four,  produced  by  division 
vertically  in  two  planes.  If  the  division  occurs  in  one  plane, 
we  have  as  a  result  small  squares  of  round  cells  known  as 


THE  BIOLOGY  OF  BACTERIA  II 

merismopedia.  In  both  these  conditions  it  frequently  hap- 
pens that  the  contiguous  sides  of  the  elements  of  packets 
become  faceted  or  straightened  against  each  other.  It  may 
happen,  too,  particularly  in  the  sarcince,  that  segmentation  is 
not  complete,  and  that  the  elements  are  larger  than  in  any 
other  class  of  cocci.  They  stain  very  readily.  Nearly  all 
the  cocci  are  non-motile,  though  Brownian  movement  may 
readily  be  observed. 

2.  The  Bacilli.     These    consist    of    rods,  having   parallel 
sides  and  being  longer  than  they  are  broad.     They  differ  in 
every  other   respect   according  to   species,  but   these   two 
characteristics  remain  to  distinguish  them.     Many  of  them 
are  motile,  others  not.     The  ends  or  poles  of  a  bacillus  may 
be  pointed,  round,  or  almost  exactly  square  and  blocked. 
They  all,  or  nearly  all,  possess  a  capsule.    Individuals  of  the 
same  species  may  differ  greatly,  according  to  whether  they 
have  been  naturally  or  artificially  grown,  and  pleomorphic 
forms  are  abundant. 

3.  The  Spirilla.     This  wavy  thread  group  is  divisible  into 
a  number  of  different  forms,  to  which  authorities  have  given 
special  names.    It  is  sufficient,  however,  to  state  that  the  two 
common  forms  are  the  non-septate   spiral  thread  (like  the 
Spirillum  Obermeier  of  relapsing  fever),  which  takes  no  other 
form  but  a  lengthened  spirillum  ;  and  the  spirillum  which 
breaks  up  into  elements  or  units,  each  of  which  appears 
comma-shaped  (like  the  cholera  bacillus).     The  degree  of 
curvature  in  the  spirilla,  of  course,  varies.     They  are  the 
least  important  of  the  lower  bacteria. 

The  Higher  Bacteria  group  includes  more  highly  organised 
members  of  the  Schizomycetes.  They  possess  filaments, 
which  may  be  branched,  and  almost  always  have  septa  and 
a  sheath.  Perhaps  the  most  marked  difference  from  the 
lower  bacteria  is  in  their  reproduction.  In  the  higher  bac- 
teria we  have  what  is  in  fact  a  flower — terminal  fructifica- 
tion by  conidia.  In  this  group  of  vegetables  we  have  the 


12  BACTERIA 

Beggiatoa,  Leptothrix,  Cladothrix,  and,  at  the  top,  the 
Streptothrix.  It  has  been  demonstrated  that  Streptothrix 
actinomycotica  and  Streptothrix  madurce  are  the  organismal 
cause,  respectively,  of  Actinomycosis  and  Madura-foot,  two 
diseases  which  have  hitherto  been  obscure. 

Pleomorphism.  This  term  designates  an  irregular  develop- 
ment of  a  species.  Different  media  and  external  conditions 
bring  about  in  protoplasm  as  susceptible  as  mycoprotein  a 
variety  of  morphological  phases.  These  may  occur  in  suc- 
cession, and  represent  different  stages  in  the  life-history  of 
a  bacterium,  or  they  may  be  involution  forms  resulting  from 
a  change  of  environment,  and  occurring  as  "  faults  "  in  the 
species.  In  the  Bacillus  coli,  B.  typhosus,  bacillus  of  Plague, 
and  B.  tuberculosis  pleomorphism  undoubtedly  occurs,  and 
is  manifest  in  the  change  of  shape.  This  is  particularly 
marked  in  old  cultures  of  the  last  named.  The  ordinary 
well-known  bacillus  may  grow  out  into  threads,  with  bul- 
bous endings,  granular  filaments,  drumsticks,  and  diplococcal 
forms.  Speaking  generally,  the  older  the  culture,  the  more 
marked  is  the  variation. 

Polymorphism  is  a  term  used  to  define  the  theory  which 
held  that  bacteria  were  one  of  the  intermediate  shapes  or 
forms  between  something  lower  and  something  higher  in  the 
vegetable  kingdom.  Neither  pleomorphism  nor  polymor-' 
phism  is  fully  understood,  and  many  bacteriologists  find 
shelter  from  both  in  the  term  involution  form.  What  we  do 
know  is  that  the  species  already  named,  for  example,  take  on 
divers  forms  when  placed  under  different  conditions. 

Composition.  From  what  we  have  seen  of  the  diet  of  micro- 
organisms, we  shall  conclude  that  in  some  form  or  other  they 
contain  the  elements  nitrogen,  carbon,  and  hydrogen.  All 
three  substances  are  combined  in  the  mycoprotein  or  proto- 
plasm of  which  the  body  of  the  microbe  consists.  This  is 
generally  homogeneous,  and  there  is  no  sign  of  a  nucleus. 
It  possesses  a  fortunate  affinity  for  aniline  dyes,  and  by  this 


THE  BIOLOGY  OF  BACTERIA  13 

means  organisms  are  stained  for  the  microscope.  Besides  the 
variable  quantity  of  nitrogen  present,  mycoprotein  may  also 
contain  various  mineral  salts.  The  uniformity  of  the  cell 
protoplasm  may  be  materially  affected  by  disintegration  and 
segmentation  due  to  degenerative  changes.  Vacuoles  also  may 
appear  from  a  like  cause,  which  it  is  necessary  to  differentiate 
from  spores.  Two  other  signs  of  degeneration  are  the  ap- 
pearance of  granules  in  the  body  of  the  cell  protoplasm 
known  as  met  achromatic  granules,  owing  to  their  different 
staining  propensities,  and  the  polar  bodies  which  are  seen  in 
some  species  of  bacteria.  Surrounding  the  mass  of  myco- 
protein, we  find  in  most  organisms  a  capsule  or  membrane 
composed,  in  part  at  least,  of  cellulose.  This  sheath  plays  a 


»;. 

V    'X 


-vi; 


NORMAL  AND  PLEON-FORMS  OF  THE  BACILLUS  OF  TUBASH. 

protective  part  in  several  ways.  During  the  adult  stage  of 
life  it  protects  the  mycoprotein,  and  holds  it  together.  At 
the  time  of  reproduction  or  degeneration  it  not  infrequently 
swells  up,  and  forms  a  viscous  hilum  or  matrix,  inside  which 
are  formed  the  new  sheaths  of  the  younger  generation.  It 
may  be  rigid,  and  so  maintain  the  normal  shape  of  the 
species,  or,  on  the  other  hand,  flexible,  and  so  adapted  to 
rapid  movement  of  the  individual. 

Here,  then,  we  have  the  major  parts  in  the  constitution  of 
a  bacillus — its  body,  mycoprotein  ;  its  capsule,  cellulose. 
But,  further  than  this,  there  are  a  number  of  additional  dis- 


14  BACTERIA 

tinctive  characteristics  as  regards  the  contents  inside  the 
capsule  which  call  for  mention.  Sulphur  occurs  in  the  Beg- 
giatoa  which  thrive  in  sulphur  springs.  Starch  is  commoner 
still.  Iron  as  oxide  or  other  combination  is  found  in  several 
species.  Many  are  highly  coloured,  though  these  are  generally 
the  "innocent"  bacteria,  in  contradistinction  to  the  disease- 
producing.  A  pigment  has  been  found  which  is  designated 
bacterio-purpurin.  According  to  Zopf,  the  colouring  agents 
of  bacteria  are  the  same  as,  or  closely  allied  to,  the  colouring 
matters  occurring  widely  in  nature.  Migula  holds  that  most 
of  the  bacterial  pigments  are  non-nitrogenous  bodies.  There 
are  a  very  large  number  of  chromogenic  bacteria,  some  of 
which  produce  exceedingly  brilliant  colours.  Among  some 
of  the  commoner  forms  possessing  this  character  are  Bacillus 
et  micrococcus  violaceus  (violet) ;  B.  ct  M.  aurantiacus 
(orange)  ;  B.  et  M.  luteus  (yellow)  ;  M.  roseus  (pink)  ;  many 
of  the  Sarcince ;  B.  aureus  (golden-yellow);  B.  fluorescens 
liquefaciens  et  non-liquefaciens  (green)  ;  B.  pyocyaneus  (green)  ; 
B.  prodigiosus  (blood-red). 

Motility.  When  a  drop  of  water  containing  bacteria  is 
placed  upon  a  slide,  a  clean  cover  glass  superimposed,  and 
the  specimen  examined  under  an  oil  immersion  lens,  various 
rapid  movements  will  generally  be  observed.  These  are  of 
four  kinds:  (i)  A  dancing  stationary  motion  known  as 
Brownian  movement.  This  is  molecular,  and  depends  in 
some  degree  upon  heat  and  the  medium  of  the  moving 
particles.  It  is  non-progressive,  and  is  well  known  in  gam- 
boge particles.  (2)  An  undulatory  serpentine  movement, 
with  apparently  little  advance  being  made.  (3)  A  rotatory 
movement,  which  in  some  water  bacilli  is  very  marked,  and 
consists  of  spinning  round,  with  sometimes  considerable 
velocity,  and  maintained  for  some  seconds  or  even  minutes. 
(4)  A  progressive  darting  movement,  by  which  the  bacillus 
passes  over  some  considerable  distance. 

The  conditions  affecting  the  motion  of  bacteria  are  but 


THE  BIOLOGY  OF  BACTERIA  15 

partly  understood.  Heating  the  slide  or  medium  accelerates 
all  movement.  A  fresh  supply  of  oxygen,  or  indeed  the 
addition  of  some  nutrient  substance,  like  broth,  will  have 
the  same  effect.  There  are  also  the  somewhat  mysterious 
powers  by  which  cells  possess  inherent  attraction  or  repulsion 
for  other  cells,  known  as  positive  and  negative  chemiotaxis. 
These  powers  have  been  observed  in  bacteria  by  Pfeiffer  and 
Ali-Cohen. 

The  essential  condition  in  the  motile  bacilli  is  the  presence 
of  flagella?      These  cilia,  or  hairy  processes,  project  from 


BACILLI,  SHOWING  FLAGELLA 

the  sides  or  from  the  ends  of  the  rod,  and  are  freely  motile 
and  elastic.  Sometimes  only  one  or  two  terminal  flagella 
are  present ;  in  other  cases,  like  the  bacillus  of  typhoid 
fever,  five  to  twenty  may  occur  all  round  the  body  of  the 
bacillus,  varying  in  length  and  size,  sometimes  being  of 
greater  length  even  than  the  bacillus  itself.  It  is  not  yet 
established  as  to  whether  these  vibratile  cilia  are  prolonga- 
tions of  capsule  only,  or  whether  they  contain  something  of 

1  A  flagellum  is  a  hair-like  process  arising  from  the  poles  or  sides  of  the 
bacillus.  It  must  not  be  confused  with  a  filament,  which  is  a  thread-like  growth 
of  the  bacillus  itself. 


1 6  BACTERIA 

the  body  protoplasm.  Migula  holds  the  former  view,  and 
states  that  the  position  of  flagella  is  constant  enough  for 
diagnostic  purposes.  They  are  but  rarely  recognisable 
except  by  means  of  special  staining  methods.  Micrococcus 
agilis  (Ali-Cohen)  is  the  only  coccus  which  has  flagella  and 
active  motion. 

Modes  of  Reproduction.  Budding,  division,  and  spore  form- 
ation are  the  three  chief  ways  in  which  Schizomycetes  and 
Saccharomycetes  (yeasts)  reproduce  their  kind.  Budding 
occurs  in  some  kinds  of  yeast,  and  would  be  classified  by 
some  authorities  under  spore  formation,  but  in  practice  it  is 
so  obviously  a  "  budding  "  that  it  may  be  so  classified.  The 
capsule  of  a  large  or  mother  cell  shows  a  slight  protrusion 
outwards  which  is  gradually  enlarged  into  a  daughter  yeast 
and  later  on  becomes  constricted  at  the  neck.  Eventually 
it  separates  as  an  individual.  The  protoplasm  of  spores  of 
yeasts  differs,  as  Hansen  has  pointed  out,  according  to  their 
conditions  of  culture. 

Division,  or  fission,  is  the  commonest  method  of  repro- 
duction. It  occurs  transversely.  A  small  indentation  occurs 
in  the  capsule,  Which  appears  to  make  its  way  slowly 
through  the  whole  body  of  the  bacillus  or  micrococcus  until 
the  two  parts  are  separate,  and  each  contained  in  its  own 
capsule.  It  has  been  pointed  out  already  that  in  the  incom- 
plete division  of  micrococci  we  observe  a  stage  precisely 
similar  to  a  diplococcus.  So  also  in  the  division  of  bacilli 
an  appearance  occurs  described  as  a  diplobacillus. 

Simple  fission  requires  but  a  short  period  of  time  to  be 
complete.  Hence  multiplication  is  very  rapid,  for  within 
half  an  hour  a  new  adult  individual  can  be  produced.  It 
has  been  estimated  that  at  this  rate  one  bacillus  will  in 
twenty-four  hours  produce  17,000,000  similar  individuals; 
or,  expressed  in  another  way,  Cohn  calculated  that  in  three 
days,  under  favourable  circumstances,  this  rate  of  increase 
would  form  a  mass  of  living  organisms  weighing  7300  tons, 


THE  BIOLOGY  OF  BACTERIA  I/ 

and  numbering  about  4772  billions.  Favourable  conditions 
do  not  occur,  fortunately,  to  allow  of  such  increase,  which,  of 
course,  can  only  be  roughly  estimated.  But  the  above  figures 
illustrate  the  enormous  fertility  of  micro-organic  life.  When 
we  remember  that  in  some  species  it  requires  10,000  or  15,000 
fully  grown  bacilli  placed  end  to  end  to  stretch  the  length 
of  an  inch,  we  see  also  how  exceedingly  small  are  the  in- 
dividuals composing  these  unseen  hosts. 

Spore  formation  may  result  in  the  production  of  germinat- 
ing cells  inside  the  capsule  of  the  bacillus,  endospores,  or  of 
modified  individuals,  arthrospores.  The  body  of  a  bacillus, 
in  which  sporulation  is  about  to  occur,  loses  its  homogeneous 
character  and  becomes  granular,  owing  to  the  appearance  of 
globules  in  the  protoplasm.  In  the  course  of  three  or  four 
hours  the  globule  enlarges  to  fill  the  diameter  of  the  rod,  and 
assumes  a  more  concentrated  condition  than  the  parent  cell. 
At  its  maturity,  and  before  its  rupture  of  the  bacillary  cap- 
sule, a  spore  is  observed  to  be  bright  and  shining,  oval  and 
regular  in  shape,  with  concentrated  contents,  and  frequently 
causing  a  local  expansion  of  the  bacillus.  In  a  number  of 
rods  lying  endwise,  these  local  swellings  produce  a  beaded 
or  varicose  appearance,  even  simulating  a  streptococcus.  In 
the  meantime  the  rod  itself  has  become  slightly  broader 
and  pale.  Eventually  it  breaks  down  by  segmentation  or 
by  swelling  up  into  a  gelatinous  mass.  The  spore  now 
escapes  and  commences  its  individual  existence.  Under 
favourable  circumstances  it  will  germinate.  The  tough  cap- 
sule gives  way  at  one  point,  generally  at  one  of  the  poles, 
and  the  spore  sprouts  like  a  seed.  In  the  space  of  about 
one  hour's  time  the  oval  refractile  cell  has  become  a  new 
bacillus.  One  spore  produces  by  germination  one  bacillus. 
Spores  never  multiply  by  fission,  nor  reproduce  themselves. 

Hueppe  has  stated  that  there  are  certain  organisms  (like 
leuconostoc,  and  some  streptococci)  which  reproduce  by  the 
method  of  arthrospores.  Defined  shortly,  this  is  simply  an 


i8 


BACTERIA 


enlargement  of  one  or  more  cell  elements  in  the  chain  which 
thus  takes  on  the  function  of  maternity.  On  either  side  of 
the  large  coccus  may  be  seen  the  smaller  ones,  which  it  is 
supposed  have  contributed  of  their  protoplasm  to  form  a 
mother  cell.  An  arthrospore  is  said  to  be  larger,  more  re- 
fractile,  and  more  resistant  than  an  ordinary  endospore. 
Many  bacteriologists  of  repute  have  declined  hitherto  to 
definitely  accept  arthrospore  formation  as  a  proved  fact. 

It  is  important  to  note  that  spore  formation  in  bacteria 
must  not  be  considered  as  a  method  of  multiplication.     The 


>    # 

VARIOUS  FORMS  OF  SPORE  FORMATION  AND  FLAGELLA 

A.  Stages  in  formation  of  spore  and  its  after  development.    B.  Spirillum  with  terminal  flagella. 

general  rule  is  undoubtedly  that  one  bacillus  produces  one 
spore,  and  one  spore  germinates  into  one  bacillus.  It  is  a  re- 
production, not  a  multiplication.  Indeed,  the  whole  process 
is  of  the  nature  of  a  resting  stage,  and  is  due  (a)  to  the 
arrival  of  the  adult  bacillus  at  its  biological  zenith,  or  (V)  to 
the  conditions  in  which  it  finds  itself  being  unfavourable 


THE  BIOLOGY  OF  BACTERIA  19 

to  its  highest  vegetative  growth,  and  so  it  endeavours  to 
perpetuate  its  species.  Most  authorities  are  probably  of  the 
latter  opinion,  though  there  is  not  a  little  evidence  for  the 
former.  Exactly  what  conditions  are  favourable  to  sporula- 
tion  is  not  known.  Nutriment  has  probably  an  intimate 
effect  upon  it.  The  temperature  must  not  be  below  16°  C., 
nor  much  above  40°  C.  Oxygen,  as  we  have  seen,  is  favour- 
able, if  not  necessary,  to  many  species,  which  will  in  cultiva- 
tion in  broth  rise  to  the  surface  and  lodge  in  the  pellicle  to 
form  their  seeds.  Moisture,  too,  is  considered  a  necessity. 

The  position  and  size  of  the  spore  are  of  considerable  use  in 
differential  diagnosis.  The  terminal  spore  of  Bacillus  tetani 
is  well  known.  It  is  rarely  seen  at  both  ends  of  the  bacillus, 
and  hence  when  poised  only  at  one  end  causes  the  "  drum- 
stick "  appearance.  In  the  bacillus  of  Quarter  Evil  the 
spore  is  generally  towards  one  end  of  the  rod  rather  than 
in  the  middle ;  in  Malignant  CEdema  the  bacillus  in  the 
blood  grows  out  into  long  threads,  and  when  such  a  thread 
sporulates  the  spore  is  also  near  one  end.  The  latter  further 
illustrates  the  fact  that  in  some  species  the  spore  is  of 
greater  diameter  than  the  mother  cell,  and  hence  dilates 
the  bacillary  capsule.  The  spores  of  anthrax  are  typical 
oval  endospores.  When  free  in  the  field  of  the  microscope, 
spores  must  be  distinguished  from  fat  cells,  micrococci, 
starch  cells,  some  kinds  of  ova,  yeast  cells,  and  other  like 
objects.  Spores  are  detected  frequently  by  their  resistance 
to  ordinary  stains  and  the  necessity  of  colouring  them  by 
special  staining  methods.  When,  however,  a  spore  has 
taken  on  the  desired  colour,  it  retains  it  with  tenacity.  In 
addition  to  their  shape,  size,  thickened  capsule,  and  staining 
characteristics,  spores  also  resist  desiccation  and  heat  in  a 
much  higher  degree  than  bacilli  not  bearing  spores.  Roux 
and  some  other  eminent  bacteriologists  suggest  that  bacteria 
should  be  classified  according  to  their  method  of  spore 
formation. 


20  BACTERIA 

THE  INFLUENCE  OF  EXTERNAL  CONDITIONS  ON 
GROWTH  OF  BACTERIA 

Nutritive  Medium.  In  the  very  earliest  days  of  the  study 
of  micro-organisms  it  was  observed  that  they  mostly  con- 
gregate where  there  is  pabulum  for  their  nourishment. 
The  reason  why  fluids  such  as  milk,  and  dead  animal  matter 
such  as  a  carcass,  and  living  tissues  such  as  a  man's  body 
contain  so  many  microbes  is  because  each  of  these  three 
media  is  favourable  to  their  growth.  Milk  affords  almost 
an  ideal  food  and  environment  for  microbes.  Its  tempera- 
ture and  constitution  frequently  meet  their  requirements. 
Dead  animal  matter,  too,  yields  a  rich  diet  for  some  species 
(saprophytes).  In  the  living  tissues  bacteria  obtain  not  only 
nutriment,  but  a  favourable  temperature  and  moisture.  Out- 
side the  human  body  it  has  been  the  endeavour  of  bacterio- 
logists to  provide  media  as  like  the  above  as  possible, 
and  containing  many  of  the  same  elements  of  food.  Thus 
the  life-history  may  be  carried  on  outside  the  body  and 
under  observation.  By  means  of  cover-glass  preparations 
for  the  microscope  we  are  able  to  study  the  form,  size, 
motility,  flagella,  spore  formation,  and  peculiarities  of  stain- 
ing, all  of  which  characters  aid  us  in  determining  to  what 
species  the  organism  under  examination  belongs.  By  means 
of  artificial  nutrient  media  we  may  further  learn  the  char- 
acters of  the  organism  in  "  pure  culture," '  its  favourable 
temperature,  its  power  or  otherwise  of  liquefaction,  the  curd- 
ling milk,  or  of  gas  production,  its  behaviour  towards 
oxygen,  its  power  of  producing  indol,  pigment,  and  chemical 
bodies,  as  well  as  its  thermal  death  point  and  resistance  to 
light  and  disinfectants.  It  is  well  known  that  under  artificial 
cultivation  an  organism  may  be  greatly  modified  in  its 
morphology  and  physiology,  and  yet  its  conformity  to  type 

1  A  "  pure  culture  "  is  a  growth  in  an  artificial  medium  outside  the  body  of 
one  species  of  micro-organism  only. 


THE  BIOLOGY  OF  BACTERIA  21 

remains  much  more  marked  than  any  degeneration  which 
may  occur. 

The  basis  of  many  of  these  artificial  media  is  broth.  This 
is  made  from  good  lean  beef,  free  from  fat  and  gristle,  which 
is  finely  minced  up  and  extracted  in  sterilised  water  (one 
pound  of  lean  beef  to  every  1000  cc.  of  water).  It  is  then 
filtered  and  sterilised.  It  will  be  understood  that  such  an 
extract  is  acid.  To  provide  peptone  beef-broth,  ten  grains  of 
peptone  and  five  grains  of  common  salt  are  added  to  every 
litre  of  acid  beef-broth.  It  is  rendered  slightly  alkaline  by  the 
addition  of  sodium  carbonate,  and  is  filtered  and  sterilised. 
Glycerine-broth  indicates  that  6  to  8  per  cent,  of  glycerine  has 
been  added  after  filtration,  glucose-broth  I  or  2  per  cent,  of 
grape-sugar.  This  latter  is  used  for  anaerobic  organisms. 
The  use  of  broth  as  a  culture  medium  is  of  great  value.  It 
is  undoubtedly  our  best  fluid  medium,  and  in  it  may  not 
only  be  kept  pure  cultures  of  bacteria  which  it  is  desired  to 
retain  for  a  length  of  time,  but  in  it  also  emulsions  and  mix- 
tures may  be  placed  preparatory  to  further  operations. 
Gelatine  is  broth  solidified  by  the  addition  of  100  grams  of 
best  French  gelatine  to  the  litre.  Its  advantage  is  twofold : 
it  is  transparent,  and  it  allows  manifestation  of  the  power  of 
liquefaction.  When  we  speak  of  a  liquefying  organism  we 
mean  a  germ  having  the  power  of  producing  a  peptonising 
ferment  which  can  at  the  temperature  of  the  room  break 
down  solid  gelatine  into  a  liquid.  Grape-sugar  gelatine  is 
made  like  grape-sugar  broth.  Agar  was  introduced  as  a 
medium  which  would  not  melt  at  25°  C,  like  gelatine,  but 
remain  solid  at  blood-heat  (37*5°  C.  ;  98*5°  F.).  It  is  a  sea- 
weed generally  obtained  in  dried  strips  from  the  Japanese 
market.  Ten  to  fifteen  grams  are  added  to  every  litre  of 
peptone-broth.  Filtration  is  slow  and  often  difficult,  and 
the  result  not  as  transparent  as  desirable.  The  former  diffi- 
culty is  avoided  by  filtering  in  the  Koch's  steamer  or  with 
a  hot-water  filter,  the  latter  by  the  addition  of  the  white  of 


22  BACTERIA 

an  egg-  Glycerine  and  grape-sugar  may  be  added  as  else- 
where. Blood  agar  is  ordinary  agar  with  fresh  sterile  blood 
smeared  over  its  surface.  Blood  serum  is  drawn  from  a  jar 
of  coagulated  horse-blood,  in  which  the  serum  has  risen  to 
the  top.  This  is  collected  in  sterilised  tubes  and 
coagulated  in  a  special  apparatus  (the  serum  in- 
spissator).  Potato  is  prepared  by  scraping  ordin- 
ary potatoes,  washing  in  corrosive  sublimate,  and 
sterilising.  They  may  then  be  cut  into  various 
shapes  convenient  for  cultivation.  Upon  any  of 
these  forms  of  solid  media  the  characteristic 
growth  of  the  organism  can  be  observed.  Of  the 
nutr^ent  elements  required,  nitrogen  is  obtained 
PREPARED  FOR  f rom  albumens  and  proteids,  carbon  from  milk- 
SUgarj  cane-sugar,  or  the  splitting  up  of  proteids  ; 
salts  (particularly  phosphates  and  salts  of  potas- 
buibofthetubesjum)  are  reacjily  obtainable  from  those  incorpor- 
ated in  the  media ;  and  the  water  which  is  required  is 
obtainable  from  the  moisture  of  the  media. 

There  are  two  common  forms  of  test-tube  culture,  viz. :  on 
the  surface  and  in  the  depth  of  the  medium.  In  the  former 
the  medium  is  sloped,  and  the  inoculating  needle  is  drawn 
along  its  surface ;  in  the  latter  the  needle  is  thrust  vertically 
downwards  into  the  depth  of  the  solid  medium.  Plate  cul- 
tures and  anaerobic  cultures  will  be  described  at  a  later 
stage.  When  the  medium  has  been  inoculated  the  culture 
is  placed  at  a  temperature  which  will  be  favourable.  Two 
standards  of  temperature  are  in  use  in  bacteriological  labora- 
tories. The  one  is  called  room  temperature,  and  varies  from 
18°  C.-2O0  C. ;  the  other  is  blood-heat,  and  varies  from  35° 
€.-38°  C.  It  is  true,  some  species  will  grow  below  18°  C., 
and  others  above  38°  C.  The  pathogenic  (disease-producing) 
bacteria  thrive  best  at  37°  C.,  and  the  non-pathogenic  at  the 
ordinary  temperature  of  the  room.  The  different  degrees 
of  temperature  are  regulated  by  means  of  incubators.  For 


STAPHYLOCOCCUS  PYOGENES  AUREUS 


INCUBATOR 

(Temperature  of  blood-heat,  registered  by  thermometer,  and  regulated 
by  thermo-regulator) 


THE  BIOLOGY  OF  BACTERIA 


the  low  temperatures  gelatine  is  chosen  ;  as  a  medium  for  the 
higher  temperatures  agar. 

Moisture  has  been  shown  to  have  a  favourable  effect  upon 
the  growth  of  microbes.  Drying  will  of  itself  kill  many 
species  (e.g.,  the  spirillum  of  cholera),  and,  other  things  being 
equal,  the  moister  a  medium  is,  the  better  will  be  the  growth 
upon  it.  Thus  it  is  that  the  growth  in  broth  is  always  more 
luxuriant  than  that  on  solid  media.  Yet  the  growth  of 


CULTURE  MEDIA  READY  FOR  INOCULATION 

Bacillus  subtilis  and  other  species  is  an  exception  to   this 
rule,  for  they  prefer  a  dry  medium. 

Temperature.  Most  bacteria  grow  well  at  room  temper- 
ature, but  they  will  grow  more  luxuriantly  and  speedily  at 
blood-heat.  The  optimum  temperature  is  generally  that  of 
the  natural  habitat  of  the  organism.  In  exceptional  cases 
growth  will  occur  as  low  as  5°  C.  or  as  high  as  70°  C. 
Indeed,  some  have  been  cooled  to  -20°  C.  and  -30°  C.,  and 
yet  retained  their  vitality,1  whereas  some  few  can  grow  at 

1  Some  pathogenic  germs  (suppuration  and  typhoid)  can  withstand  freezing 
for  weeks. 


24  BACTERIA 

60-70°   C.     These  latter  are  termed  thermophilic  bacteria. 
The  average  thermal  death-point  is  at  or  about  50°  C. 

Light  acts  as  an  inhibitory  or  even  germicidal  agent.     This 
fact  was  first  established  by  Downes  and  Blunt  in  a  memoir 


INOCULATING  NEEDLES 

Plantinum  wire  fused  into  glass  handles 

to  the  Royal  Society  in  1877.  They  found  by  exposing 
cultures  to  different  degrees  of  sunlight  that  thus  the  growth 
of  the  culture  was  partially  or  entirely  prevented,  being  most 
damaged  by  the  direct  rays  of  the  sun,  although  diffuse  day- 
light acted  prejudicially.  Further,  these  same  investigators 
proved  that  of  the  rays  of  the  spectrum  which  acted  inimi- 
cally  the  blue  and  violet  rays  acted  most  bactericidally,  next 
to  the  blue  being  the  red  and  orange-red  rays.  The  action 
of  light,  they  explain,  is  due  to  the  gradual  oxidation  which 
is  induced  by  the  sun's  rays  in  the  presence  of  oxygen. 
Duclaux,  who  worked  at  this  question  at  a  later  date,  con- 
cluded that  the  degree  of  resistance  to  the  bactericidal  in- 
fluence of  light  which  some  bacteria  possess  might  be  due 
to  difference  in  species,  difference  jn  culture  media,  and 
difference  in  the  degrees  of  intensity  of  light.  Tyndall  tested 
the  growth  of  organisms  in  flasks  exposed  to  air  and  light 
on  the  Alps,  and  found  that  sunlight  inhibited  the  growth 
temporarily.  A  large  number  of  experimenters  in  Europe 
and  England  have  worked  at  this  fascinating  subject  since 


PASTEUR'S  LARGE  INCUBATOR  FOR  CULTIVATION  AT  ROOM  TEMPERATURE 


THE  BIOLOGY  OF  BACTERIA  2$ 

1877,  and  though  many  of  their  results  appear  contradictory, 
we  may  be  satisfied  to  adopt  the  following  conclusions  re- 
specting the  matter : 

(1)  Sunlight  has  a  deleterious  effect  upon  bacteria,  and  to 
a  less  extent  on  their  spores. 

(2)  This  inimical    effect    can   be    produced    by  light  irre- 
spective of  rise  in  temperature. 

(3)  The  ultra-violet  rays  are  the  most  bactericidal,  and  the 
infra-red  the  least  so,  which  indicates  that  the  phenomenon 
is  due  to  chemical  action. 

(4)  The  presence  of  oxygen  and  moisture  greatly  increases 
this  action. 

(5)  The  sunlight  acts  prejudicially  upon  the  culture  me- 
dium, and  thereby  complicates  the  investigation  and  after- 
growth. 

(6)  The  time  occupied  in  the  bactericidal  action  depends 
upon  the  heat  of  the  sun  and  the  intrinsic  vitality  of  the 
organism. 

(7)  With  regard  to  the  action  of  light  upon  pathogenic 
organisms,  some  results  have  recently  been  obtained  with 
Bacillus  typhosus.     Janowski  maintains  that  direct  sunlight 
exerts  a  distinctly  depressing  effect  on  typhoid  bacilli.     At 
present  more  cannot  be  said  than  that  sunlight  and  fresh 
air  are  two  of  the  most  powerful  agents  we  possess  with 
which  to  combat  pathogenic  germs. 

A  very  simple  method  of  demonstrating  the  influence  of 
light  is  to  grow  a  pure  culture  in  a  favourable  medium, 
either  in  a  test-tube  or  upon  a  glass  plate,  and  then  cover 
the  whole  with  black  paper  or  cloth.  A  little  window  may 
then  be  cut  in  the  protective  covering,  and  the  whole  exposed 
to  the  light.  Where  it  reaches  in  direct  rays  it  will  be  found 
that  little  or  no  growth  has  occurred  ;  where,  on  the  other 
hand,  the  culture  has  been  in  the  dark,  abundant  growth 
occurs.  In  diffuse  light  the  growth  is  merely  somewhat  in- 
hibited. It  has  been  found  that  the  electric  light  has  but 


26  BACTERIA 

little  action  upon  bacteria,  though  that  which  it  has  is  similar 
to  sunlight.  Recent  experiments  with  the  Rontgen  rays 
have  given  negative  results. 

In  1890  Koch  stated  that  tubercle  bacilli  were  killed  after 
an  exposure  to  direct  sunlight  of  from  a  few  minutes  to 
several  hours.  The  influence  of  diffuse  light  would  obviously 
be  much  less.  Professor  Marshall  Ward  has  experimented 
with  the  resistant  spores  of  Bacillus  anthracis  by  growing 
these  on  agar  plates  and  exposing  to  sunlight.  From  two  to 
six  hours'  exposure  had  a  germicidal  effect. 

It  should  be  remembered  that  several  species  of  sea-water 
bacteria  themselves  possess  powers  of  phosphorescence. 
Pfluger  was  the  first  to  point  out  that  it  was  such  organisms 
which  provided  the  phosphorescence  upon  decomposing  wood 
or  decaying  fish.  To  what  this  light  is  due,  whether  capsule, 
or  protoplasm,  or  chemical  product,  is  not  yet  known.  The 
only  facts  at  present  established  are  to  the  effect  that  certain 
kinds  of  media  and  pabulum  favour  or  deter  phosphor- 
escence. 

Desiccation.  A  later  opportunity  will  occur  for  considera- 
tion of  the  effect  of  drying  upon  bacteria.  Here  it  is  only 
necessary  to  say  that,  other  things  being  equal,  drying 
diminishes  virulence  and  lessens  growth. 

Oxygen.  Pasteur  was  the  first  to  lay  emphasis  upon  the 
effect  which  free  air  had  upon  micro-organisms.  He  classi- 
fied them  according  to  whether  they  grew  in  air,  aerobic,  or 
whether  they  flourished  most  without  it,  anaerobic.  Some 
have  the  faculty  of  growing  with  or  without  the  presence  of 
oxygen,  and  are  designated  as  facultative  aerobes  or  anaer- 
obes. As  regards  the  .cultivation  of  anaerobic  germs,  it  is 
only  necessary  to  say  here  that  hydrogen,  nitrogen,  or  car- 
bonic acid  gas  may  be  used  in  place  of  oxygen,  or  they  may 
be  grown  in  a  medium  containing  some  substance  which 
will  absorb  the  oxygen. 

Modes  of  Bacterial  Action.     In    considering   the  specific 


THE  BIOLOGY  OF  BACTERIA  2/ 

action  of  micro-organisms,  it  is  desirable,  in  the  first  place, 
to  remember  the  two  great  functional  divisions  of  saprophyte 
and  parasite.  A  saprophyte  is  an  organism  that  obtains  its 
nutrition  from  dead  organic  matter.  Its  services,  of  what- 
ever nature,  lie  outside  the  tissues  of  living  animals.  Its  life 
is  spent  apart  from  a  "  host."  K  parasite,  on  the  other  hand, 
lives  always  at  the  expense  of  some  other  organism  which  is 
its  host,  in  which  it  lives  and  upon  which  it  lives.  There  is  a 
third  or  intermediate  group,  known  as  "  facultative,"  owing 


METHOD  OF  PRODUCING  HYDROGEN  BY  KIPP'S  APPARATUS  FOR 
CULTIVATION  OF  ANA£ROBES  (See  page  139) 

to  their  ability  to  act  as  parasites  or  saprophytes,  as  the 
exigencies  of  their  life-history  may  demand. 

The  saprophytic  organisms  are,  generally  speaking,  those 
which  contribute  most  to  the  benefit  of  man,  and  the  parasitic 
the  reverse,  though  this  statement  is  only  approximately  true. 
In  their  relation  to  the  processes  of  fermentation,  decompos- 
ition, nitrification,  etc.,  we  shall  see  how  great  and  invaluable 
is  the  work  which  saprophytic  microbes  perform.  Their  re- 
sult depends,  in  nearly  all  cases,  upon  the  organic  chemical 
constitution  of  the  substances  upon  which  they  are  exerting 


28 


BACTERIA 


their  action,  as  well  as  upon  the  varieties  of  bacteria  them- 
selves. Nor  must  it  be  understood  that  the  action  of  sapro- 
phytes is  wholly  that  of  breaking  down  and  decomposition. 
As  a  matter  of  fact,  some  of  their  work  is,  as  we  shall  see, 
of  a  constructive  nature  ;  but,  of  whichever  kind  it  is,  the 
result  depends  upon  the  organism  and  its  environment. 
This,  too,  may  be  said  of  the  pathogenic  species,  all  of 
which  are  in  a  greater  or  less  degree  parasitic.  It  is  well 
known  how  various  are  the  constitutions  of  man, 
how  the  bodies  of  some  persons  are  more  resistant 
than  those  of  others,  and  how  the  invading  microbe 
will  find  different  receptions  according  to  the  con- 
stitution and  idiosyncrasy  of  the  body  which  it 
attacks.  Indeed,  even  after  invasion  the  infectivity 
of  the  special  disease,  whatever  it  happens  to  be, 
will  be  materially  modified  by  the  tissues.  When 
we  come  to  turn  to  the  micro-organisms  which 
are  pathogenic  parasites  we  shall  further  have  to 
keep  clear  in  our  minds  that  their  action  is  double 
and  complex,  and  not  single  or  simple.  In  the 
first  place,  we  have  an  infection  of  the  body  due 
ANAEROBIC  to  tke  bacteria  themselves.  It  may  be  a  general 
(Buckner'sTube)and  widespread  infection,  as  in  anthrax,  where  the 
with  Pyrogaiiic bacilli  pass,  in  the  blood  or  lymph  current,  to 

Solution  in  Bulb.  r^uuj  '-u  u 

each  and  every  part  of  the  body ;  or  it  may  be  a 
comparatively  local  one,  as  in  diphtheria,  where  the  in- 
vader remains  localised  at  the  site  of  entrance.  But,  be 
that  as  it  may,  the  micro-organisms  themselves,  by  their 
own  bodily  presence,  set  up  changes  and  perform  functions 
which  may  have  far-reaching  effects.  It  is  obvious  that 
the  wider  the  distribution  the  wider  is  the  area  of  tissue 
change,  and  vice  versa.  Yet  there  is  something  of  far 
greater  importance  than  the  mere  presence  of  bacteria  in 
human  or  animal  tissues  ;  for  the  secondary  action  of  disease- 
producing  germs — and  possibly  it  is  present  in  all  bacteria 


THE   BIOLOGY  OF  BACTERIA 


29 


— is  due  to  their  poisonous  products,  or  toxins,  as  they 
have  been  termed.  These  may  be  of  the  nature  of  fer- 
ments, and  they  become  diffused  throughout  the  body, 
whether  the  bacteria  themselves  occur  locally  or  generally. 
They  may  bring  about  very  slight  and  even  imperceptible 
changes  during  the  course  of  the  disease,  or  they  may  kill 
the  patient  in  a  few  hours.  Latterly  bacteriologists  have 
come  to  understand  that  it  is  not  so  much  the  presence 
of  organisms  which  is  injurious  to  man  and  other  animals, 
as  it  is  their  products  which  cause  the  mischief  ;  and  the 
amount  of  toxic  product  bears  no  known  proportion  to  the 
degree  of  invasion  by  the  bacteria.  The  various  and  widely 
differing  modes  of  action  in  bacteria  are  therefore  dependent 
upon  these  three  elements:  the  tissues  or  medium,  the  bac- 
teria, and  the  products  of  the  bacteria  ;  and  in  all  organismal 
processes  these  three  elements  act  and  react  upon  each  other. 

A  word  may  be  said  here  respecting  the  much-discussed 
question  of  species  in  bacteria.  A  species  may  be  defined  as 
"  a  group  of  individuals  which,  however  many  characters  they 
share  with  other  individuals,  agree  in  presenting  one  or  more 
characters  of  a  peculiar  and  hereditary  kind  with  some  certain 
degree  of  distinctness."  '  Now,  as  regards  bacteria,  there  is 
no  doubt  that  separate  species  occur  and  tend  to  remain  as 
separate  species.  It  is  true,  there  are  many  variations,  due  in 
large  measure  to  the  medium  in  which  the  organisms  are 
growing, — variations  of  age,  adaptation,  nutrition,  etc., — yet 
the  different  species  tend  to  remain  distinct.  Involution 
forms  occur  frequently,  and  degeneration  invariably  modifies 
the  normal  appearance.  But  because  of  the  occurrence  of 
these  morphological  and  even  pathological  differences  it  must 
not  be  argued  that  the  demarcation  of  species  is  wholly 
arbitrary. 

Means  of  Sterilisation.  As  this  term  occurs  frequently  in 
even  a  book  of  this  untechnical  nature,  and  as  it  is  expressive 

1  G.  J.  Romanes,  Darwin  and  After  Darwin,  vol.  ii.,  231. 


3O  BACTERIA 

of  an  idea  which  must  always  be  present  to  the  mind  of  the 
bacteriologist,  it  may  be  desirable  to  make  some  passing 
allusion  to  it. 

Chemical  substances,  perfect  filtration,  and  heat  are  the 
three  means  at  our  command  in  order  to  secure  germ-free 
conditions  of  apparatus  or  medium.  The  first  two,  though 
theoretically  admissible,  are  practically  seldom  used,  the 
former  of  the  two  because  the  addition  of  chemical  substances 
annuls  or  modifies  the  operation,  the  latter  of  the  two  on 
account  of  the  great  practical  difficulties  in  securing  perfec- 
tion. Hence  in  the  investigation  involved  in  bacteriological 
research  heat  is  the  common  sterilising  agent.  A  temperat- 
ure of  70°  C.  (158°  F.)  will  kill  all  bacilli;  even  58°  C.  will 
kill  most  kinds.  Boiling  at  100°  C.  (212°  F.)  for  three  min- 
utes will  kill  anthrax  spores,  and  boiling  for  thirty  to  sixty 
minutes  will  kill  all  bacilli  and  all  spores.  This  differ- 
ence in  the  thermal  death-point  between  bacilli  and  their 
spores  enables  the  operator  to  obtain  what  are  called  "  pure 
cultures  "  of  a  desired  bacillus  from  its  spores  which  may  be 
present.  For  example,  if  a  culture  contains  spores  of  an- 
thrax and  is  contaminated  with  micrococci,  heating  to  70° 
C.  (158°  F.)  will  kill  all  the  micrococci,  but  will  not  affect  the 
spores  of  anthrax,  which  can  then  grow  into  a  pure  culture 
of  anthrax  bacilli.  Fractional  or  discontinuous  sterilisation 
depends  on  the  principle  of  heating  to  the  sterilising  point 
for  bacilli  (say  70°  C.)  on  one  day,  which  will  kill  the  bacilli, 
but  leave  the  spores  uninjured.  But  by  the  following  day 
the  spores  will  have  germinated  into  bacilli,  and  a  second 
heating  to  70°  C.  will  kill  them  before  they  in  their  turn 
have  had  time  to  sporulate.  Thus  the  whole  will  be  sterilised, 
though  at  a  temperature  below  boiling. 

Successful  sterilisation,  therefore,  depends  upon  killing 
both  bacteria  and  their  spores,  and  nothing  short  of  that 
can  be  considered  as  sterilisation.  The  following  methods 
are  those  generally  used  in  the  laboratory.  For  dry  heat 


THE   BIOLOGY  OF  BACTERIA 


(which  is  never  so  injurious  to  organisms  as  moist  heat )  *  : 
(a)  the  Bunsen  burner,  in  the  flame  of  which  platinum 
needles,  etc.,  are  sterilised  ;  (b)  hot-air  chamber,  in  which 
flasks  and  test-tubes  are  heated  to  a  temperature  of  150-170° 
C.  for  half  an  hour.  For  moist 
heat :  (c)  boiling,  for  knives  and  in- 
struments ;  (d)  Koch 's  steam  steri- 
liser, by  means  of  which  a  crate  is 
slung  in  a  metal  cylinder,  at  the 
bottom  of  which  the  water  is 
boiled  ;  (e)  the  autoclave,  which  is 
the  most  rapid  and  effective  of  all 
the  methods.  This  is  in  reality  a 
Koch  steriliser,  but  with  apparatus 
for  obtaining  high  pressure.  The 
last  two  (d,  e)  are  used  for  sterilis- 
ing the  nutriment  media  upon 
which  bacteria  are  cultivated  out- 
side the  body.  Blood  serum 
would,  however,  coagulate  at  a 
temperature  over  60°  C.  (124°  F.), 
and  hence  a  special  steriliser  has 
been  designed  to  carry  out  frac- 
tional sterilisation  daily  for  a  week 
at  about  55°  €.-58°  C. 

The  Association  of  Organisms. 
At  a  later  stage  we  shall  have  an 
opportunity  of  discussing  symbi- 
osis and  allied  conditions.  Here 
it  is  only  necessary  to  draw  attention  to  a  fact  that  is  rapidly 

1  It  will  be  observed  that  there  is  a  marked  difference  between  the  effects  of 
dry  heat  and  moist  heat.  Moist  heat  is  able  to  kill  organisms  much  more  readily 
than  dry,  owing  to  its  penetrating  effect  on  the  capsule  of  the  bacillus.  Dry 
heat  at  140°  C.  (284°  F.),  maintained  for  three  hours,  is  necessary  to  kill  the 
resistant  spores  of  Bacillus  anthracis  and  B.  subtilis,  but  moist  heat  at  fifty 
degrees  less  will  have  the  same  effect.  It  is  from  data  such  as  these  that  in 


KOCH'S  STEAM  STERILISER 


32  BACTERIA 

becoming  of  the  first  importance  in  bacteriology.  When 
species  were  first  isolated  in  pure  culture  it  was  found  that 
they  behaved  somewhat  differently  under  differing  circum- 
stances. This  modification  in  function  has  been  attributed 
to  differences  of  environment  and  physical  conditions. 
Whilst  it  is  true  that  such  external  conditions  must  have  a 
marked  effect  upon  such  sensitive  units  of  protoplasm  as 
bacteria,  it  has  recently  been  proved  that  one  great  reason 
why  modification  occurs  in  pure  artificial  cultures  is  that  the 
species  has  been  isolated  from  amongst  its  colleagues  and 
doomed  to  a  separate  existence.  One  of  the  most  abstruse 
problems  in  the  immediate  future  of  the  science  of  bacterio- 
logy is  to  learn  what  intrinsic  characters  there  are  in  species 
or  individuals  which  act  as  a  basis  for  the  association  of 
organisms  for  a  specific  purpose.  Some  bacteria  appear  to 
be  unable  to  perform  their  regular  function  without  the  aid 
of  others.  An  example  of  such  association  is  well  illustrated 
in  the  case  of  tetanus,  for  it  has  been  shown  that  if  the 
bacilli  and  spores  of  tetanus  alone  obtain  entrance  to  a 
wound  the  disease  may  not  follow  the  same  course  as  when 
with  the  specific  organism  the  lactic-acid  bacillus  or  the 
common  organisms  of  suppuration  or  putrefaction  also  gain 
entrance.  There  is  here  evidently  something  gained  by  as- 
sociation. Again,  the  virulence  of  other  bacteria  is  also  in- 
creased by  means  of  association.  The  Bacillus  coli  is  an 
example,  for,  in  conjunction  with  other  organisms,  this  ba- 
cillus, although  normally  present  in  health  in  the  aliment- 
ary canal,  is  able  to  set  up  acute  intestinal  irritation,  and 

laboratories  and  in  disinfecting  apparatus  moist  heat  is  invariably  preferred  to 
dry  heat.  For  with  the  latter  such  high  temperatures  would  be  required  that 
they  would  damage  the  articles  being  disinfected.  Koch  states  the  following 
figures  for  general  guidance  :  Dry  heat  at  a  temperature  of  120°  C.  (248°  F.) 
will  destroy  spores  of  mould  fungi,  micrococci,  and  bacilli  in  the  absence  of 
their  spores  ;  for  the  spores  of  bacilli  140°  C.  (284°  F.),  maintained  for  three 
hours,  is  necessary  ;  moist  heat  at  100°  C.  (212°  F.)  for  fifteen  minutes  will  kill 
bacilli  and  their  spores. 


THE  BIOLOGY  OF  BACTERIA  33 

various  changes  in  the  body  of  an  inflammatory  nature.  It 
is  not  yet  possible  to  say  in  what  way  or  to  what  degree  the 
association  of  bacteria  influences  their  role.  That  is  a  pro- 
blem for  the  future.  But  whilst  we  have  examples  of  this  as- 
sociation in  streptococcus  and  the  bacillus  of  diphtheria,  B. 
coli  and  yeasts,  tetanus  and  putrefactive  bacteria,  Diplococ- 
cus  pneumonia  and  streptococcus,  and  association  amongst 
the  various  suppurative  organisms,  we  cannot  doubt  that 
there  is  an  explanation  to  be  found  here  of  many  hitherto 
unsolved  results  of  bacterial  action.  This  is  the  place  in 
which  mention  should  also  be  made  of  higher  organisms  as- 
sociated for  a  specific  purpose  with  bacteria.  There  is  some 
evidence  to  support  the  belief  that  some  of  the  Leptotricheae 
(Crenothrix,Beggiatoa,  Leptothrix,etc.)and  the  Cladotricheae 
(Cladothrix)  perform  a  preliminary  disintegration  of  organic 
matter  before  the  decomposing  bacteria  commence  their 
labours.  This  occurs  apparently  in  the  self-purification  of 
rivers,  as  well  as  in  polluted  soils. 

Antagonism  of  Bacteria.  Study  of  the  life-history  of 
many  of  the  water  bacteria  will  reveal  the  fact  that  they 
can  live  and  multiply  under  conditions  which  would  at  once 
prove  fatal  to  other  species.  Some  of  these  water  organisms 
can  indeed  increase  and  multiply  in  distilled  water,  whereas 
it  is  known  that  other  species  cannot  even  live  in  distilled 
water,  owing  to  the  lack  of  pabulum.  Thus  we  see  that 
what  is  favourable  for  one  species  may  be  the  reverse  for 
another. 

Further,  we  shall  have  opportunity  of  observing,  when 
considering  the  bacteriology  of  water  and  sewage,  that  there 
is  in  these  media  in  nature  a  keen  struggle  for  the  survival 
of  the  fittest  bacteria  for  each  special  medium.  In  a  carcass 
it  is  the  same.  If  saprophytic  bacteria  are  present  with 
pathogenic,  there  is  a  struggle  for  the  survival  of  the  latter. 
Now  whilst  this  is  in  part  due  to  a  competition  owing  to  a 
limited  food  supply  and  an  unlimited  population,  as  occurs 


34  BACTERIA 

in  other  spheres,  it  is  also  due  in  part  to  the  inimical  in- 
fluence of  the  chemical  products  of  the  one  species  upon  the 
life  of  the  bacteria  of  the  other  species.  Moreover,  in  one 
culture  medium,  as  Cast  has  pointed  out,  two  species  will 
often  not  grow.  When  Pasteur  found  that  exposure  to  air 
attenuated  his  cultures,  he  pointed  out  that  it  was  not  the  air 
per  se  that  hindered  his  growth,  but  it  was  the  introduction 
of  other  species  which  competed  with  the  original.  The 
growth  of  the  spirillum  of  cholera  is  opposed  by  Bacillus 
pyogenes  fcetidus.  B.  anthracis  is,  in  the  body,  opposed  by 
either  B.pyocyaneus  or  Streptococcus  erysipelatis,  and  yet  it  is 
aided  in  its  growth  by  B.  prodigiosus.  B.  aceti  is,  under 
certain  circumstances,  antagonistic  to  B.  coli  commums. 

In  several  of  the  most  recent  of  the  admirable  reports  of 
Sir  Richard  Thorne  issued  from  the  Medical  Department  of 
the  Local  Government  Board,  we  have  the  record  of  a  series 
of  experiments  performed  by  Dr.  Klein  into  this  question  of 
the  antagonism  of  microbes.  From  this  work  it  is  clearly 
demonstrated  that  whatever  opposition  one  species  affords  to 
another  it  is  able  to  exercise  by  means  of  its  poisonous  pro- 
perties. These  are  of  two  kinds.  There  is,  as  is  now  widely 
known,  the  poisonous  product  named  the  toxin,  into  which 
we  shall  have  to  inquire  more  in  detail  at  a  later  stage. 
There  is  also  in  many  species,  as  Dr.  Klein  has  pointed  out, 
a  poisonous  constituent  or  constituents  included  in  the  body 
protoplasm  of  the  bacillus,  and  which  he  therefore  terms  the 
intracellular  poison.  Now,  whilst  the  former  is  different  in 
every  species,  the  latter  may  be  a  property  common  to 
several  species.  Hence  those  having  a  similar  intracellular 
poison  are  antagonistic  to  each  other,  each  member  of  such 
a  group  being  unable  to  live  in  an  environment  of  its  own 
intracellular  poison.  Further,  it  has  been  suggested  that 
there  are  organisms  possessing  only  one  poisonous  property, 
namely,  their  toxin — for  example,  the  bacilli  of  tetanus  and 
diphtheria — whilst  there  are  other  species,  as  above,  possess- 


THE  BIOLOGY  OF  BACTERIA  35 

ing  a  double  poisonous  property,  an  intracellular  poison  and 
a  toxin.  In  this  latter  class  would  be  included  the  bacilli  of 
Anthrax  and  Tubercle. 

Reference  has  been  made  to  the  associated  work  of  higher 
vegetable  life  and  bacteria.  The  converse  is  also  true.  'Just 
as  we  have  bacterial  diseases  affecting  man  and  animals,  so 
also  plant  life  has  its  bacterial  diseases.  Wakker,  Prillieux, 
Erwin  Smith,  and  others  have  investigated  the  pathogenic 
conditions  of  plants  due  to  bacteria,  and  though  this  branch 
of  the  science  is  in  its  very  early  stages,  many  facts  have 
been  learned.  Hyacinth  disease  is  due  to  a  flagellated 
bacillus.  The  wilt  of  cucumbers  and  pumpkins  is  a  common 
disease  in  some  districts  of  the  world,  and  may  cause  wide- 
spread injury.  It  is  caused  by  a  white  microbe  which  fills 
the  water-ducts.  Wilting  vines  are  full  of  the  same  sticky 
germs.  Desiccation  and  sunlight  have  a  strongly  prejudicial 
effect  upon  these  organisms.  Bacterial  brozvn-rot  of  potatoes 
and  tomatoes  is  another  plant  disease  probably  due  to  a 
bacillus.  The  bacillus  passes  down  the  interior  of  the  stem 
into  the  tubers,  and  brown-rots  them  from  within.  There 
is  another  form  of  brown-rot  which  affects  cabbages.  It 
blackens  the  veins  of  the  leaves,  and  a  woody  ring  which 
is  formed  in  the  stem  causes  the  leaves  to  fall  off.  This 
also  is  due  to  a  micro-organism,  which  gains  entrance  through 
the  water-pores  of  the  leaf,  and  subsequently  passes  into  the 
vessels  of  the  plants.  It  multiplies  by  simple  fission,  and 
possesses  a  flagellum. 

There  can  be  no  doubt  that  these  complex  biological  pro- 
perties of  association  and  antagonism,  as  well  as  the  parasitic 
growth  of  bacteria  upon  higher  vegetables,  are  as  yet  little 
understood,  and  we  may  be  glad  that  any  light  is  being  shed 
upon  them.  In  the  biological  study  of  soil  bacteria  in 
particular  may  we  expect  in  the  future  to  find  examples  of 
association,  even  as  already  there  are  signs  that  this  is  so 
in  certain  pathogenic  conditions.  In  the  alimentary  canal,  on 


36  BACTERIA 

the  other  hand,  and  in  conditions  where  organic  matter  is 
greatly  predominating,  we  may  expect  to  see  further  light 
on  the  subject  of  antagonism. 

Attenuation  of  Virulence  or  Function.  It  was  pointed  out 
by  some  of  the  pioneer  bacteriologists  that  the  function  of 
bacteria  suffered  under  certain  circumstances  a  marked 
diminution  in  power.  Later  workers  found  that  such  a 
change  might  be  artificially  produced.  Pasteur  introduced 
the  first  method,  which  was  the  simple  one  of  allowing 
cultures  to  grow  old  before  sub-culturing.  Obviously  a  pure 
culture  cannot  last  for  ever.  To  maintain  the  species  in 
characteristic  condition  it  is  necessary  frequently  to  sub- 
culture upon  fresh  media.  If  this  simple  operation  be 
postponed  as  long  as  possible  consistent  with  vitality,  and 
then  performed,  it  will  be  found  that  the  sub-culture  is 
attenuated,  i.  e.,  weakened.  Another  mode  is  to  raise  the 
pure  culture  to  a  temperature  approaching  its  thermal  death 
point.  A  third  way  of  securing  the  same  end  is  to  place  it 
under  disadvantageous  external  circumstances,  for  example 
a  too  alkaline  or  too  acid  medium.  A  fourth,  but  rarely 
necessary,  method  is  to  pass  it  through  the  tissues  of  an 
insusceptible  animal.  Thus  we  see  that,  whilst  the  favour- 
able conditions  which  we  have  considered  afford  full  scope 
for  the  growth  and  performance  of  functions  of  bacteria, 
we  are  able  by  a  partial  withdrawal  of  these,  short  of  that 
ending  fatally,  to  modify  the  character  and  strength  of 
bacteria.  In  future  chapters  we  shall  have  opportunity  of 
observing  what  can  be  done  in  this  direction. 


CHAPTER  II 

BACTERIA  IN  WATER 

IN  entering  upon  a  consideration  of  such  a  common  article 
of  use  as  water,  we  shall  do  well  to  describe  in  some 
detail  the  process  by  which  we  systematically  investigate  the 
bacteriology  of  a  water,  or,  indeed,  of  any  similar  fluid  sus- 
pected of  bacterial  pollution. 

The  collection  of  samples,  though  it  appears  simple 
enough,  is  sometimes  a  difficult  and  responsible  under- 
taking. Complicated  apparatus  is  rarely  necessary,  and 
fallacies  will  generally  be  avoided  by  observing  two  direc- 
tions. In  the  first  place,  the  sample  should  be  chosen  as 
representative  as  possible  of  the  real  substance  or  conditions 
we  wish  to  examine.  Some  authorities  advise  that  it  is 
necessary  to  allow  the  tap  to  run  for  some  minutes  pre- 
vious to  collecting  the  sample;  but  if  we  desire  to  examine 
for  lead  chemically  or  for  micro-organisms  in  the  pipes 
biologically,  then  such  a  proceeding  would  be  injudicious.1 
Hence  we  must  use  common  sense  in  the  selection  and 
obtaining  of  a  sample,  following  this  one  guide,  namely, 
to  collect  as  nearly  as  possible  a  sample  of  the  exact  water 
the  quality  of  which  it  is  desired  to  learn.  In  the  second 
place,  we  must  observe  strict  bacteriological  cleanliness  in 
all  our  manipulations.  This  means  that  we  must  use  only 
sterilised  vessels  or  flasks  for  collecting  the  sample,  and  in 
the  manipulation  required  we  must  be  extremely  careful 

1  Water  from  a  house  cistern  is  rarely  a  fair  sample.  It  should  be  taken 
fr«:m  the  main.  If  taken  from  a  stream  or  still  water,  the  collecting  bottle 
should  be  held  about  a  foot  below  the  surface  before  the  stopper  is  removed. 

37 


38  BACTERIA 

to  avoid  any  pollution  of  air  or  any  addition  to  the 
organisms  of  the  water  from  unsterilised  apparatus.  A 
flask  polluted  in  only  the  most  infinitesimal  degree  will 
entirely  vitiate  all  results. 

Accompanying  the  sample  should  be  a  more  or  less  full 
statement  of  its  source.  There  can  be  no  doubt  that,  in 
addition  to  a  chemical  and  bacteriological  report  of  a  water, 
there  should  also  be  made  a  careful  examination  of  its 
source.  This  may  appear  to  take  the  bacteriologist  far 
afield,  and  in  point  of  fact,  as  regards  distance,  this  may 
be  so.  But  until  he  has  seen  for  himself  what  "  the  gather- 
ing-ground "  is  like,  and  from  what  sources  come  the  feeding 
streams,  he  cannot  judge  the  water  as  fairly  as  he  should  be 
able  to  do.  The  configuration  of  the  gathering-ground,  its 
subsoil,  its  geology,  its  rainfall,  its  relation  to  the  slopes 
which  it  drains,  the  nature  of  its  surface,  the  course  of  its 
feeders,  and  the  absence  or  presence  of  cultivated  areas,  of 
roads,  of  houses,  of  farms,  of  human  traffic,  of  cattle  and 
sheep — all  these  points  must  be  noted,  and  their  influence, 
direct  or  indirect,  upon  the  water  carefully  borne  in  mind. 

When  the  sample  has  been  duly  collected,  sealed,  and  a 
label  affixed  bearing  the  date,  time,  and  conditions  of  col- 
lection and  full  address,  it  should  be  transmitted  with  the 
least  possible  delay  to  the  laboratory.  Frequently  it  is 
desirable  to  pack  the  bottles  in  a  small  ice  case  for  transit. 
On  receipt  of  such  a  sample  of  water  the  examination  must  be 
immediately  proceeded  with,  in  order  to  avoid,  as  far  as  possi- 
ble, the  fallacies  arising  from  the  rapid  multiplication  of  germs. 
Even  in  almost  pure  water,  at  the  ordinary  temperature  of  a 
room,  Frankland  found  organisms  multiplied  as  follows : 

No.  of  Germs 
Hours.  per  cc. 

o ... 1,073 

6 6,028 

24.  . 7,262 

48 48, 100 


BACTERIA   IN   WATER  39 

Another  series  of  observations  revealed  the  same  sort  of 
rapid  increase  of  bacteria.  On  the  date  of  collection  the 
micro-organisms  per  cc.  in  a  deep-well  water  (in  April)  were 
seven.  After  one  day's  standing  at  room  temperature  the 
number  had  reached  twenty-one  per  cc.  After  three  days 
under  the  same  conditions  it  was  495,000  per  cc.  At  blood- 
heat  the  increase  would,  of  course,  be  much  greater,  as  a 
higher  temperature  is  more  favourable  to  multiplication. 
But  this  would  depend  upon  the  degree  of  impurity  in  the 
water,  a  pure  water  decreasing  in  number  on  account  of  the 
exhaustion  of  the  pabulum,  whereas,  for  the  first  few  days 
at  all  events,  an  organically  polluted  water  would  show  an 
enormous  increase  in  bacteria. 

Furthermore,  it  is  desirable  to  remember  that  organisms, 
in  an  ordinary  water,  do  not  continue  to  increase  indefinitely. 
There  is  a  limit  to  all  things,  even  to  numbers  in  bacte- 
riology. Cramer,  of  Zurich,  examined  the  water  of  the  Lake 
after  it  had  been  standing  for  different  periods,  with  the 
following  results:  — 

Hours  and  Days  of  No.  of  Micro-organisms 

Examination.  per  cc.1 

o  hours 143 

24     "      12,457 

3  days 323,543 

8     "     233,452 

17     "     17,436 

70     "     2,500 

The  writer's  own  experience  is  entirely  in  agreement  with 
this  cessation  of  multiplication  at  or  about  the  end  of  a  week, 
and  the  later  decline. 

Method  of  Examination.  At  the  outset  of  a  systematic 
study  of  a  water  it  is  well  to  observe  its  physical  characters. 
The  colour,  if  any,  should  be  noted.  Suspended  matter  and 

1  The  cubic  centimetre  (cc.)  is  a  convenient  standard  of  fluid  measurement 
constantly  recurring  in  bacteriology.  It  is  equal  to  16-20  drops,  and  28  cc. 
equal  one  fluid  ounce. 


40  BACTERIA 

deposit  may  indicate  organic  or  inorganic  pollution.  If 
abundant  or  conspicuous,  a  microscopic  examination  of  the 
sediment  may  be  made.  The  reaction,  whether  acid,  neutral, 
or  alkaline,  must  be  tested,  and  the  exact  temperature  taken. 
Any  and  every  fact  will  help  us,  perhaps  not  so  much  to 
determine  the  contents  of  the  water  as  to  interpret  rightly 
the  facts  we  deduce  from  the  entire  examination. 

At  the  beginning  of  the  bacteriological  work  the  water 
should  be  examined  by  means  of  the  gelatine  plate  method. 
This  consists  in  drawing  up  into  a  fine  sterilised  pipette  a 
small  quantity  of  the  water  and.  introducing  it  thereby  into 
a  test-tube  of  melted  gelatine  at  a  temperature  below  40°  C.1 


LEVELLING  APPARATUS  FOR  KOCH'S  PLATE 

It  will  depend  upon  the  apparent  quality  of  the  water  as  to 
the  exact  quantity  introduced  into  the  gelatine  ;  about  .5  or 
.1  of  a  cubic  centimetre  is  a  common  figure.  The  stopper  is 
then  quickly  replaced  in  the  test-tube,  and  the  contents  gently 
mixed  more  or  less  equally  to  distribute  the  one-tenth  cubic 
centimetre  throughout  the  melted  gelatine.  A  sterilised  sheet 
of  glass  (4  inches  by  3)  designated  a  KocJis  plate  is  now 
taken  and  placed  upon  the  stage  of  a  levelling  apparatus, 
which  holds  iced  water  in  a  glass  jar  under  the  stage. 
The  gelatine  is  now  poured  out  over  the  glass  plate,  and  by 

1  The  gelatine  is  reduced  to  liquid  form  by  heating  in  a  water-bath.  Before 
inserting  the  suspected  water  it  is  essential  that  the  gelatine  be  under  40°  C.  or 
thereabouts,  in  order  not  to  approach  the  thermal  death-point  of  any  bacteria. 


BACTERIA    IN    WATER  41 

means  of  a  sterilised  rod  stroked  into  a  thin,  even  film  all 
over  the  glass.  It  is  then  covered  with  a  bell-jar  and  left 
at  rest  to  set.  The  level  stage  prevents  the  gelatine  running 
over  the  edge  of  the  plate ;  the  iced  water  under  the  stage 


MOIST  CHAMBER  IN  WHICH  KOCH'S  PLATES  ARK  INCUBATED 

expedites  the  setting  of  the  gelatine  into  a  fixed  film.  When 
it  is  thus  set  the  plate  is  placed  upon  a  small  stand  in  a 
moist  chamber,  and  the  whole  apparatus  removed  to  the 
room  temperature  incubator.  A  moist  chamber  is  a  glass 
dish,  in  which  some  filter  paper,  soaked  with  corrosive  sub- 
limate, is  inserted,  and  the  dish  covered  with  a  bell-jar.  By 
this  means  the  risks  of  pollution  are  minimized,  and  moisture 
maintained.  In  all  cases  at  least  two  plates  must  be  pre- 
pared of  the  same  sample  of  water,  and  it  is  often  advisable 
to  make  several.  They  may  be  made  with  different  media 
for  different  purposes,  and  with  different  quantities  of  water, 
though  the  same  method  of  procedure  is  adopted.  In  a 
highly  polluted  water  extremely  small  quantities  would  be 
taken,  and,  vice  versa,  in  pure  water  a  large  quantity. 

When  we  come  to  discuss  the  relation  of  disease  organ- 
isms to  water,  particularly  those  causing  typhoid  fever,  we 
shall  learn  that  they  are  both  scarce  and  intermittent.  This 
point  has  been  dwelt  upon  frequently  by  Dr.  Klein,  and  it 
is  clear  that  such  a  state  of  things  greatly  enhances  the  diffi- 
culties in  detecting  such  bacteria,  and  he  has  proposed  a 
simple  procedure  by  which  the  difficulty  of  finding  the 
Bacillus  typhosus  in  a  large  body  of  water  may  be  met. 


BACTERIA 


One  or  two  thousand  cubic  centimetres  of  the  water  under 
examination  are  passed  through  a  sterilised  Berkefeld  filter 
by  means  of  siphon  action  or  an  air-pump.  The  candle  of 
the  filter  retains  on  its  outer  surface  all,  or  nearly  all,  the 


HOT  AIR  STERILISER 

For  the  Sterilization  of  Glass  Apparatus,  etc. 

particulate  matter  contained  in  the  water.  The  matter  thus 
retained  on  this  outer  surface  is  brushed  by  means  of  a 
sterile  brush  into  10  or  20  cc.  of  sterilised  water.  Thus  we 
have  all  the  organisms  contained  in  two  litres  of  the  water 
reduced  into  10  cc.  of  water.  From  this,  so  to  speak,  con- 


BACTERIA    IN    WATER 


43 


centrated  emulsion  of  the  bacteria  of  the  original  water, 
phenol-gelatine  plates  or  Eisner  plates  (both  acid  media) 
may  be  readily  made.  In  this  way  we  not  only  catch  many 
bacteria  which  would  evade  us  if  we  were  content  with  the 
examination  merely  of  a  few  drops  of  the  water,  but  we 
eliminate  by  means  of  the  acid  those  common  water  bacteria, 

O 


c 


like  Bacillus  fluorescens  liquefaciens,  which  so  greatly  confuse 
the  issue. 

In  the  course  of  two  or  three  days  the  film  of  gelatine 
on  the  plate  becomes  covered  with  colonies  of  germs,  and  the 
next  step  is  to  examine  these  quantitatively  and  qualitat- 
ively. We  may  here  insert  a  simple  scheme  by  which  this 
may  be  most  fully  and  easily  accomplished : — 

I.    Naked-Eye  Observation  of  the  Colonies.     By  this  means 


44  BACTERIA 

at  the  very  outset  certain  facts  may  be  obtained,  viz.,  the  size, 
elevation,  configuration,  margin,  colour,  grouping,  number, 
and  kinds  of  colonies,  all  of  which  facts  are  of  importance, 
and  assist  in  final  diagnosis.  Moreover,  in  the  case  of  gel- 
atine plates  (it  is  otherwise  in  agar)  one  is  able  to  observe 
whether  or  not  there  is  present  what  is  termed  liquefaction 
of  the  gelatine.  Some  organisms  produce  in  their  develop- 
ment a  peptonizing  ferment  which  breaks  down  gelatine  into 
a  fluid  condition.  Many  have  not  this  power,  and  hence 
the  characteristic  is  used  as  a  diagnostic  feature. 

2.  Microscopic  Examination  of  Colonies,  which  confirms  or 
corrects  that  which  has  been  observed   by  the  naked   eye. 
Fortunately  some  micro-organisms  when  growing  in  colon- 
ies produce  cultivation  features  which  are  peculiar  to  them- 
selves (especially  is    this    so   when    growing    in    test-tube 
cultures),  and    in    the  early  stages  of  such  growths  a  low 
power   of   the    microscope    or    magnifying    glass    facilitates 
observation. 

3.  Make  cover-glass  preparations:    (a)    unstained-^-"  the 
hanging  drop  "  ;  (b)  stained — single  stains,  like  gentian-violet, 
methyl  blue,  fuchsin,  carbol  fuchsin,  etc. ;   double  stains — 
Gram's  method,  Ziehl-Neelsen's  method,  etc. 

This  third  part  of  the  investigation  is  obviously  to  prepare 


\ 


THE  HANGING  DROP 

specimens  for  the  microscope.  "  The  hanging  drop  "  is  a 
simple  plan  for  securing  the  organisms  for  microscopic 
examination  in  a  more  or  less  natural  condition.  A  hollow 
ground  slide,  which  is  a  slide  with  a  shallow  depression  in  it, 
is  taken,  and  a  small  ring  of  vaseline  placed  round  the  edge 


BACTERIA    IN    WATER 


45 


of  the  depression.  Upon  the  under  side  of  a  clean  cover- 
glass  is  placed  a  drop  of  pure  water,  and  this  is  inoculated 
with  the  smallest  possible  particle  taken  from  one  of  the 
colonies  of  the  gelatine  plate  on  the  end  of  a  sterilised 
platinum  wire.  The  cover-glass  is  then  placed  upon  the 
ring  of  vaseline,  and  the  drop  hangs  into  the  space  of  the 
depression.  Thus  is  obtained  a  view  of  the  organisms  in  a 
freely  moving  condition,  if  they  happen  to  be  motile  bac- 
teria. As  a  matter  of  practice  the  hollow  slide  may  be 
dispensed  with,  and  an  ordinary  slide  used. 

With  regard  to  staining,  it  will  be    undesirable   here  to 
dwell  at  length  upon  the  large  number  of  methods  which 


DRYING  STAGE  FOR  FIXING  FILMS 

have  been  adopted.  The  "single  stain  "  may  be  shortly  men- 
tioned. It  is  as  follows  :  A  clean  cover-glass  is  taken  (cleaned 
with  nitric  acid  and  alcohol,  or  bichromate  of  potash  and 
alcohol),  and  a  drop  of  pure  sterilised  water  placed  upon  it. 
This  is  inoculated  with  the  particle  of  a  colony  on  the  end 
of  a  platinum  needle,  and  a  scum  is  produced.  The  film  is 
now  "  fixed  "  by  slowly  drying  it  over  a  flame.  When  the 
scum  is  thus  dried,  a  drop  of  the  selected  stain  (say  gentian, 
violet)  is  placed  over  the  scum  and  allowed  to  remain  for 
varying  periods  :  sarcince  about  thirty  seconds  ;  for  many  of 
the  bacilli  three  or  four  minutes.  It  is  then  washed  off  with 
clean  water,  dried,  and  mounted  in  Canada  balsam.  The 
organisms  will  now  appear  under  the  microscope  as  violet 
in  colour,  and  will  thus  be  clearly  seen. 

The    "  double  staining"   is  adopted  when  we  desire  to 


46  BACTERIA 

stain  the  organisms  one  colour  and  the  tissue  in  which  they 
are  situated  a  contrast  colour.  Some  of  the  details  of  these 
methods  are  mentioned  in  the  Appendix. 

4.  Sub-culture.     The  plate  method  was  really  introduced 
by  Koch   in  order  to  facilitate  isolation  of  species.     In  a 
flask  it  is  impossible  to  isolate  individual  species,  but  when 
the  growth  is  spread  over  a  comparatively  large  area,  like 
a  plate,  it  is  possible  to  separate  the  colonies,  and  this  being 
done   by  means  of   a   platinum  wire,  the    colonies   may  be 
replanted  in  fresh  media  ;  that  is  to  say,  a  sub-culture  may 
be  made,  each  organism  cultivated  on  its  favourite  soil,  and 
its  manner  of  life  closely  watched.      We  have  already  men- 
tioned the  chief  media  which  are  used  in  the  laboratory,  and 
in  an  investigation  many  of  these  would  be  used,  and  thus 
pure  cultures  would  be  obtained.      Let   us  suppose  that  a 
water  contains  six  kinds  of  bacteria.     On  the  plate  these 
six   kinds  would   show   themselves    by   their  own    peculiar 
growth.      Each  would  then  be  isolated  and  placed  in  a  sep- 
arate tube,  on  a  favourite  medium,  and  at  a  suitable  tem- 
perature.    Thus  each  would   be  a  pure  culture ;    i.   e.,  one 
and  only  one,  species  would  be  present  in  each  of  the  six  tubes. 
By  this  simple  means  an  organism  can  be,  we  say,  cultivated, 
in  the  same  sort  of  way  as  in  floriculture.      From  day  to 
day  we  can  observe  the  habits  of  each  of  our  six  species, 
and  probably  at  an  early  stage  of  their  separated  existences 
we  should  be  able  to  diagnose  what  species  of  bacteria  we 
had  found  in  the  water.    If  not,  further  microscopic  examin- 
ation could  be  made,  and,  if  necessary,  secondary   or  ter- 
tiary sub-cultures. 

5.  Inoculation   of  Animals.      It    may    be    necessary    to 
observe  the  action  of  supposed  pathogenic  organisms  upon 
animals.     This  is  obviously  a  last  resource,  and  any  abuse 
of  such  a  process  is  strictly  limited  by  law.      As  a  matter  of 
fact,  an  immense  amount  of  bacteriological  investigation  can 
be   carried  on  without   inoculating  animals  ;     but,  strictly 


BACTERIA   IN    WATER 


47 


speaking,  as  regards  many  of  the  pathogenic  bacteria,  this 
test  is  the  most  reliable  of  all.  Nor  would  any  responsible 
bacteriologist  be  justified  in  certifying  a  water  as  healthy  for 
consumption  by  a  large  community  if  he  was  in  doubt  as  to 
the  disease-producing  action  of  certain  contained  organisms. 
By  working  through  some  such  scheme  as  the  above  we 
are  able  to  detect  what  quantity  and  species  of  organisms, 
saprophytic  or  parasitic,  a  water  or  similar  fluid  contains. 


TYPES  OF  LIQUEFACTION  OF  GELATINE 

For,  observe  what  information  we  have  gained.  We  have 
learned  the  form  (whether  bacillus,  micrococcus,  or  spirillum), 
size,  consistence,  motility,  method  of  grouping,  and  staining 
reactions  of  each  micro-organism  ;  the  characters  of  its  culture, 
colour,  composition,  presence  or  absence  of  liquefication  or 
gas  formation,  its  rate  of  growth,  smell,  or  reaction;  and 
lastly,  when  necessary,  the  effect  that  it  has  upon  living 
tissues.  Here,  then,  are  ample  data  for  arriving  at  a  satis- 
factory conclusion  respecting  the  qualitative  estimation  of 
the  suspected  water. 


48  BACTERIA 

As  to  to  the  quantitative  examination,  that  is  fulfilled  by 
counting  the  number  of  colonies  which  appear,  say  by  the 
third  and  fourth  day,  upon  the  gelatine  plates.  Each  colony 
has  arisen,  it  is  assumed,  from  one  individual,  so  that  if  we 
count  the  colonies,  though  we  do  not  thereby  know  how 
many  organisms  we  have  upon  our  plate,  we  do  know 
approximately  how  many  organisms  there  were  when  the 
plate  was  first  poured  out,  which  are  the  figures  we  require, 
and  which  can  at  once  be  multiplied  and  returned  as  so 
many  organisms  per  cubic  centimetre.  There  is,  unfort- 
unately, at  present  no  exact  standard  to  which  all  bacterio- 
logists may  refer. 

Miquel  and  Crookshank  have  suggested  standards  which 
allow  "  very  pure  water  "  to  contain  up  to  100  micro-organ- 
isms per  cc.  Pure  water  must  not  contain  more  than  1000, 
and  water  containing  up  to  100,000  bacteria  per  cc.  is  con- 
taminated with  surface  water  or  sewage.  Mace  gives  the 
following  table  : 

Very  pure  water o-        10  bacteria  per  cc. 

Very  good  water 20-      100       "  " 

Good  water 100-      200       "  " 

Passable  (mediocre)  water 200-      500       "  " 

Bad  water 500-  1,000       "  " 

Very  bad  water 1,000-10,000  and  over     " 

Koch  first  laid  emphasis  on  the  quantity  of  bacteria  present 
as  an  index  of  pollution,  and  whilst  different  authorities 
have  all  agreed  that  there  is  a  necessary  quantitative  limit, 
it  has  been  so  far  impossible  to  arrive  at  one  settled  standard 
of  permissible  impurity. 

Besson  adopts  the  standard  suggested  by  Miquel,  and,  on 
the  whole,  French  bacteriologists  follow  suit.  They  also 
agree  with  him,  generally  speaking,  in  not  placing  much 
emphasis  upon  the  numerical  estimation  of  bacteria  in  water. 
In  Germany  and  England  it  is  the  custom  to  adopt  a  stricter 
limit.  Koch  in  1893  fixed  100  bacteria  per  cc.  as  the  maxi- 
mum number  of  bacteria  which  should  be  present  in  a 


BACTERIA    IN    WATER 


49 


properly   filtered    water.       Hence    the    following    has    been 
recognised  more  or  less  as  the  standard  : 

o-  100      bacteria  per  cc.    =a.  good  potable  water. 

loo-  500  "      =a  suspicious  water. 

500-1000  or  more  "  =a  water  which  should  have 

further  filtration  before 
being  used  for  drinking 
purposes. 

The  personal  view  of  the  writer  after  some  experience  of 
water  examination  would  favour  a  standard  of  "  under  500  " 
being  a  potable  water,  if  the  500  were  of  a  nature  indicating 
neither  sewage  pollution  nor  disease.  Miquel  holds  that  not 
more  than  ten  different  species  of  bacteria  should  be  present 
in  a  drinking  water,  and  such  is  a  useful  standard.  The 
presence  of  rapidly  liquefying  bacteria  associated  with  sewage 
or  surface  pollution  would,  even  though  present  in  fewer 


WOLFHUGEL'S  COUNTER 

numbers  than  a  standard,  condemn  a  water.  Thus  it  will 
be  seen  that  it  is  impossible  to  judge  alone  by  the  numbers 
unless  they  are  obviously  enormously  high. 

When  we  are  counting  colonies  upon  a  Koch's  plate, 
WolfhugeVs  counter  may  be  used.  This  is  a  thin  plate  of 
glass  a  size  larger  than  Koch's  plates,  and  upon  it  are 
scratched  squares,  each  square  being  divided  into  nine 
smaller  squares.  The  Wolfhiigel  plate  is  superimposed 


5O  BACTERIA 

upon  the  Koch's  plate,  and  the  colonies  counted  in  one  little 
square  or  set  of  squares  and  multiplied. 

By  using  flat,  shallow,  circular  glass  dishes,  generally  known 
as  Petris  dishes,  instead  of  Koch's  plates,  much  manipulation 
and  time  is  saved,  and,  on  the  whole,  less  risk  of  pollution 
occurs.  Moreover,  these  are  easily  carried  about  and  trans- 


PETRI'S  DISH 

ferred  from  place  to  place.  When  counting  colonies  in  a 
Petri's  dish  it  is  sufficient  to  divide  the  circle  into  eight  equal 
divisions,  and  counting  the  colonies  in  the  average  divisions, 
multiply  and  reduce  to  the  common  denominator  of  one 
cc.  For  example,  if  the  colonies  of  the  plate  appear  to  be 
distributed  fairly  uniformly  we  count  those  in  one  of  the 
divisions.  They  reach,  we  will  suppose,  the  figure  of  60; 
60  X  8=480  micro-organisms  in  the  amount  taken  from  the 
suspected  water  and  added  to  the  melted  gelatine  from 
which  the  plate  was  made.  This  amount  was  .25  cc.  There- 
fore we  estimate  the  number  of  micro-organisms  in  the  sus- 
pected water  as  60  X  8=480  X  4=1920  m.-o.  per  cc.,  which 
is  over  standard  by  about  1500.  A  water  might  then  be 
condemned  upon  its  quantitative  examination  alone  or  qual- 
itative alone,  or  both.  If  the  quantity  were  even  that  of  an 
artesian  well,  say  4-10  m.-o.  per  cc.,  but  those  four  or  ten 
were  all  Bacillus  typhosus,  it  would  clearly  be  condemned  on 
its  quality,  though  quantitatively  it  was  an  almost  pure 
water.  If,  on  the  contrary,  the  water  contained  10,000  m.-o. 
per  cc.,  and  none  of  them  disease-producing,  it  would  still  be 
condemned  on  the  ground  that  so  large  a  number  of  organ- 
isms indicated  some  kind  of  organic  pollution  to  supply 
pabulum  for  so  many  organisms  to  live  in  one  cc.  of  the  water. 
It  is  not  the  number  per  se  which  condemns.  The  large 


BACTERIA    IN  WATER  51 

number  condemns  because  it  indicates  probable  pollution 
with  surface  water  or  sewage  in  order  to  supply  pabulum 
for  so  many  bacteria  per  cc. 

It  should  always  be  remembered  that  a  chemical  report 
and  a  bacteriological  report  should  assist  each  other.  The 
former  is  able  to  tell  us  the  quantity  of  salts  and  condition 
of  the  organic  matter  present ;  the  latter  the  number  and 
quality  of  micro-organisms.  Neither  can  take  the  place  of 
the  other  and,  generally  speaking,  both  are  more  or  less  use- 
less until  we  can  learn,  by  inspection  and  investigation  of  the 
source  of  the  water,  the  origin  of  the  organic  matter  or  con- 
tamination. Hence  a  water  report  should  contain  not  only 
a  record  of  physical  characters,  of  chemical  constituents,  and 
of  the  presence  or  absence  of  micro-organisms,  injurious  and 
otherwise,  but  it  should  also  contain  information  obtained  by 
personal  investigation  of  the  source.  Only  thus  can  a  reason- 
able opinion  be  expected.  Moreover,  it  is  generally  only 
possible  to  form  an  accurate  judgment  of  a  water  from 
watching  its  history,  that  is,  not  from  one  examination  only, 
but  from  a  series  of  observations.  A  water  yielding  a  steady 
standard  of  bacterial  contents  is  a  much  more  satisfactory 
water,  from  every  point  of  view,  than  one  which  is  unstable, 
one  month  possessing  500  bacteria  per  cc.  and  another 
month  5000.  It  is  obvious  that  rainfall  and  drought,  soil 
and  trade  effluents,  will  have  their  influence  in  materially 
affecting  the  bacterial  condition  of  a  water. 

It  is  perhaps  scarcely  necessary  to  add  that  we  have  not 
in  the  above  account  of  the  examination  of  water  included 
all,  or  nearly  all,  the  various  methods  adopted  for  acquiring 
a  knowledge  of  the  bacterial  contents  of  the  water.  Many  of 
these  are  of  too  detailed  and  technical  a  nature  to  enter  into 
here.  Three  points,  however,  we  may  touch  upon.  In  the 
first  place,  as  we  have  said,  the  particulate  matter  out  of  a 
large  body  of  water  should  be  concentrated  in  a  small 
quantity.  Accordingly  it  has  become  the  custom  to  pass 


BACTERIA 


J 


2OOO  or  3000  cc.  of  the  suspected  water  through  a  Berkefeld 
filter.  When  this  has  been  accomplished,  by  means  of  a 
sterile  brush  the  particulate  matter  on  the 
candle  of  the  filter  is  brushed  off  into  10  or 
15  cc.  of  sterilised  water.  This  simple  ar- 
rangement is  analogous  to  the  use  of  gravity 
or  centrifugal  methods  of  securing  the  solid 
matter  in  milk.  The  smaller  quantity  of 
water  is  then  readily  examined,  and  scanty 
germs  more  readily  detected.  A  second 
point  elaborating  the  scheme  of  water  exam- 
ination is  the  choice  of  media  for  sub-cultur- 
ing.  Mere  examination  on  gelatine  is  not 
sufficient.  Even  in  making  the  primary 
/!  plate  cultivations  it  is  well  to  vary  the  media 
— agar,  carbol-gelatine,  Eisner,  etc.  But 
when  colonies  have  appeared  upon  these 
plates  it  is  important  to  sub-culture  with 
accuracy  and  good  judgment  upon  all  or  any 
media — gelatine,  agar,  broth,  potato,  milk, 
blood  serum,  glucose  agar,  glycerine  agar, 
BERKEFELD  FILTER  etc.— that  will  reveal  the  real  characters  of 
in  Position  for  Fiitra-  the  bacteria  present.  A  method  proposed 

tion  of  Water  to  t         -n     •  r  r»i         •  i  TA    i  /      •  •  i 

be  Examined.  by  rrotessor  bhendan  Delepme  is  to  place 
some  of  the  suspected  water  in  sterilised 
test-tubes  without  further  treatment,  and  incubate  at  37° 
C.  for  twelve  or  eighteen  hours,  and  then  plate  out  and 
estimate  the  number  of  bacteria  as  in  the  ordinary  course. 
"  In  polluted  water,  containing  an  excess  of  organic  matter," 
he  says,  "  an  extremely  rapid  multiplication  of  bacteria  is 
observed.  In  unpolluted  water,  containing  only  water  bacteria 
and  a  very  small  amount  of  organic  matter,  very  little  or  no 
multiplication  takes  place,  and  the  growth  of  the  water  bac- 
teria liquefying  gelatine  is  checked  to  a  remarkable  extent." 
Thirdly,  by  none  of  these  methods  should  we  be  able  to 


APPARATUS  FOR  FILTERING  WATER  TO  FACILITATE  ITS  BACTERIOLOGICAL 

EXAMINATION 


BACTERIA   IN    WATER  53 

isolate  anaerobic  bacteria,  and  to  furnish  a  complete  report 
these  also  must  receive  careful  attention. 

The  Bacteriology  of  Water.  In  many  natural  waters  there 
will  be  found  varied  contents  even  in  regard  to  flora  alone  : 
algce,  diatoms,  spirogyrcz,  desmids,  and  all  sorts  of  vegetable 
detritus.  Many  of  these  organisms  are  held  responsible  for 
divers  disagreeable  tastes  and  odours.  The  colour  of  a  water 
may  also  be  due  to  similar  causes.  Dr.  Garrett,  of  Chelten- 
ham, has  recorded  the  occurrence  of  redness  of  water  owing 
to  a  growth  of  Crenothrix  polyspora,  and  many  other  similar 
cases  make  it  evident  that  not  unfrequently  great  changes  may 
be  produced  in  water  by  contained  microscopic  vegetation. 

With  the  exception  of  water  from  springs  and  deep  wells, 
all  unfiltered  natural  waters  contain  numbers  of  bacteria. 
The  actual  number  roughly  depends  upon  the  amount  of  or- 
ganic pabulum  present,  and  upon  certain  physical  conditions 
of  the  water.  As  we  have  already  seen,  bacteria  multiply  with 
enormous  rapidity.  In  some  species  multiplication  does  not 
appear  to  depend  on  the  presence  of  much  organic  matter, 
and,  indeed,  some  can  live  and  multiply  in  sterilised  water: 
Micrococcus  aquatilis  and  Bacillus  erythrosportis.  Again, 
others  depend  not  upon  the  quantity  of  organic  matter,  but 
upon  its  quality.  And  frequently  in  a  water  of  a  high  de- 
gree of  organic  pollution  it  will  be  found  that  bacteria  have 
been  restrained  in  their  development  by  the  competition  of 
other  species  monopolising  the  pabulum.  Probably  at  least 
one  hundred  different  species  of  non-pathogenic  organisms 
have  been  isolated  from  water.  Some  species  are  constantly 
occurring,  and  are  present  in  almost  all  natural  waters. 
Amongst  such  are  B.  liquefaciens,  B.  fluorescent  liq.,  B.  fluor- 
escens  non-liquefaciens,  B.  termo,  B.  aquatilis,  B.  ubiquitus, 
and  not  a  few  micrococci,  etc.  Percy  Frankland  1  collected 
water  from  various  quarters  at  various  times  and  seasons, 
and  some  of  his  results  may  here  be  added  : 

1  Micro-organisms  in  Water  (1894). 


54 


BACTERIA 


RIVER  THAMES  WATER  COLLECTED  AT  HAMPTON 

Number  of  Micro-organisms  Obtained  from  i  cc.  of  Water. 


MONTH. 

1886. 

1887. 

1888. 

January 

4.5  ooo 

30,800 

92,000 

February  

15,800 

6,700 

40,000 

March 

II  415 

3O  QOO 

66  ooo 

April   .  .  . 

12  250 

52,IOO 

13  ooo 

May.. 

4,8OO 

2,IOO 

I,  QOO 

Tune 

8  300 

2  2OO 

o  CQO 

Tulv 

1  OOO 

2  5OO 

i  070 

August 

6  100 

7  200 

3,000 

September  

8,400 

l6,7OO 

1,740 

October 

8  600 

6  700 

I     JOQ 

November 

56  ooo 

81  ooo 

II  700 

December    .  . 

63  ooo 

19  ooo 

10  600 

Again,  another  example  : 

RIVER  LEA  WATER  COLLECTED  AT  CHINGFORD 

Number  of  Micro-organisms  Obtained  from  i  cc.  of  Water, 


MONTH. 

1886. 

1887. 

1888. 

January  

3Q  300 

37,7OO 

31  ooo 

February 

20  600 

7  QOO 

26  ooo 

March.  

Q  O25 

24  ooo 

63  ooo 

April 

7  300 

I   33O 

84  ooo 

May.. 

2,050 

2,2OO 

1,124 

June 

4  7OO 

12  2OO 

7  OOO 

Tulv 

5  4.00 

12  3OO 

2   IQO 

August                

4  ^OO 

5.3OO 

2  OOO 

September  

3,7OO 

Q,2OO 

1,670 

October     .                        ... 

6  4.OO 

7  6OO 

2  3IO 

November     .                    

12  700 

27  ooo 

57  5OO 

December  

I2I,OOO 

II,  OOO 

4  4OO 

"  During  the  summer  months  these  waters  are  purest  as 
regards  micro-organisms,  this  being  due  to  the  fact  that 
during  dry  weather  these  rivers  are  mainly  composed  of 
spring  water,  whilst  at  other  seasons  they  receive  the  wash- 
ings of  much  cultivated  land." — Frankland. 


BACTERIA   IN  WATER  55 

Prausnitz  has  shown  that  water  differs,  as  would  be  ex- 
pected, according  to  the  locality  in  the  stream  at  which 
examination  is  made.  His  investigations  were  made  from 
the  river  Isar  before  and  after  it  receives  the  drainage  of 

Munich: 

No.  of  Colonies 
per  cc. 

Above  Munich 531 

Near  entrance  of  principal  sewer 227,369 

13  kilometres  from  Munich 9,m 

22  "  "  "      4,796 

33          "  "     2,378 

Professor  Percy  Frankland  also  points  out  how  the  river 
Dee  affords  another  example,  even  more  perfect,  of  pollution 
and  restoration  repeated  several  times  until  the  river  be- 
comes almost  bacterially  pure. 

We  cannot  here  enter  more  fully  into  the  many  conditions 
of  a  water  which  affect  its  bacterial  content  than  to  say  that 
it  varies  considerably  with  its  source,  at  different  seasons, 
and  under  different  climatic  conditions.  An  enormous  in- 
crease will  occur  if  the  sediment  is  disturbed,  and  conversely 
sedimentation  and  subsidence  during  storage  will  greatly 
diminish  the  numbers  of  bacteria.  Sand  filtration,  plus  a 
"  nitrifying  layer,"  will  remove  more  than  90  per  cent,  of  the 
bacteria.  Sea- water  contains  comparatively  few  bacteria, 
and  the  deeper  the  water  and  the  farther  it  is  from  shore  so 
much  less  will  be  the  bacterial  pollution. 

THE  CHIEF  DISEASE  ORGANISMS  FOUND   IN  WATER 

We  will  now  consider  several  of  the  more  important  dis- 
ease-producing bacteria  found  in  water. 

Bacillus  Typhosus  (Eberth-Gaffky).  In  1 880-81  Eberth 
announced  the  discovery  of  this  bacillus  in  cases  of  clinical 
enteric  fever.  In  1884  it  was  first  cultivated  outside  the 
body  by  Gaffky.  Since  then  other  organisms  have  been  held 


56  BACTERIA 

responsible  for  the  causation  of  enteric  (or  typhoid)  fever. 
In  1885  tne  B-  c°ti  communis  was  recognised,  and  it  has  been 
a  matter  of  great  debate  amongst  bacteriologists  as  to  how 
far  these  two  organisms  are  the  same  species,  and  the 
typhoid  germ  merely  a  higher  evolution  of  the  B.  coli.  The 
differentiating  signs  between  them  will  be  referred  to  shortly. 
Bacteriologists  generally  regard  the  Eberth-Gaffky  bacillus 
as  the  specific  cause  of  the  disease,  though  complete  proof 
is  still  wanting. 

Microscopic  Characters  (in  pure  culture).  Rods,  2-4  /*  long, 
.5  fj.  broad,  having  round  ends.  Sometimes  threads  are  ob- 
servable, being  10  ^  in  length.  In  the  field  of  the  microscope 
the  bacilli  differ  in  length  from  each  other,  but  are  all  the 


BACTERIA  OF  TYPHOID  FEVER 

same  thickness  approximately.  Round  and  oval  cells  con- 
stantly occur  even  in  pure  culture,  and  many  of  these  shorter 
forms  of  typhoid  are  identical  in  morphology  with  some  of 
the  many  forms  of  Bacillus  coli.  There  are  no  spores. 
Motility  is  marked  ;  indeed,  in  young  culture  it  is  the  most 
active  pathogenic  germ  we  know.  The  small  forms  dart 
about  with  extreme  rapidity  ;  the  longer  forms  move  in  a 
vermicular  manner.  Its  powers  of  movement  are  due  to 
some  five  to  twenty  flagella  of  varying  length,  some  of  them 
being  much  longer  than  the  bacillus  itself,  though,  owing  to 
the  swelling  of  the  bacillus  under  flagellum-staining  methods, 


BACTERIA    IN    WATER  57 

it  is  difficult  to  gauge  this  exactly.  The  flagella  are  terminal 
and  lateral,  and  are  elastic  and  wavy.  Their  active  con- 
traction produces  an  evident  current  in  the  field  of  the 
microscope. 

Cultures.  This  organism  may  be  isolated  from  ulcerated 
Peyer's  patches  in  the  intestine,  from  the  liver,  the  spleen, 
and  the  mesenteric  glands.  Owing  to  the  mixture  of  bacteria 
found  elsewhere,  it  is  generally  best  to  isolate  it  from  the 
spleen.  The  whole  spleen  is  removed,  and  a  portion  of  its 
capsule  seared  with  a  hot  iron  to  destroy  superficial  organ- 
isms. With  a  sterilised  knife  a  small  cut  is  made  into  the 
substance  of  the  organ,  and  by  means  of  a  sterilised  platinum 
wire  a  little  of  the  pulp  is  removed  and  traced  over  the  sur- 
face of  agar.  Agar  reveals  a  growth  in  about  twenty-four 
hours  at  37°  C.,  which  is  the  favourite  temperature.  A  grey- 
ish, moist,  irregular  growth  appears,  but  it  is  invariably  at- 
tached to  the  track  of  the  inoculating  needle.  On  gelatine 
the  growth  is  much  the  same,  but  its  irregular  edge  is,  if  any- 
thing, more  apparent.  There  is  no  liquefaction  and  no  gas 
formation.  On  plates  of  gelatine  the  colonies  appear  large 
and  spreading,  with  jagged  edges.  The  whole  colony  ap- 
pears raised  and  almost  limpet-shaped,  with  delicate  lines 
passing  over  its  surface.  There  is  an  appearance  under  a  low 
power  of  transparent  iridescence.  The  growth  on  potato  is 
termed  "  invisible,"  and  is  of  the  nature  of  a  potato-coloured 
pellicle,  which  looks  moist,  and  may  at  a  late  stage  become 
a  light  brown  in  colour,  particularly  if  the  potato  is  alkaline. 
Milk  is  a  favourable  medium,  and  is  rendered  slightly  acid. 
No  coagulation  takes  place.  Broth  is  rendered  turbid. 

Micro-pathology.  Typhoid  fever  is  an  infiltration  and  co- 
agulation, necrosis,  and  ulceration  of  the  Peyer's  patches  in 
the  small  intestine  of  man.  The  mesenteric  glands  show  the 
same  features,  except  that  there  is  no  ulceration.  The  spleen 
is  enlarged,  and  contains  the  germs  of  the  disease  in  almost 
a  pure  culture.  The  bacillus  is  present  in  the  intestinal  con- 


BACTERIA 


tents  and  excreta,  particularly  so  when  the  Peyer's  glands 
have  commenced  ulceration.  In  the  blood  of  the  general 
circulation  the  bacillus  is  not  demonstrable,  except  in  very 
rare  instances.  Typhoid  fever  is  not,  like  anthrax,  a  blood 
disease. 

COMPARATIVE  FEATURES  OF  BACILLUS  TYPHOSUS  AND 

B.  COLI 


B.    TYPHOSUS 

Morphology  :  Cylindrical  bacillus  2.4 
>M,  unequal  lengths  ;  some  fila- 
ments. 

Plagella  :  Long,  wavy,  spiral,  and 
very  numerous  ;  movement  very 
active. 

On  Gelatine  and  Agar  :  Angular,  ir- 
regular, raised  colonies ;  slow 
growth  ;  translucent ;  medium  re- 
mains clear. 

In  Gelatine  :  In  ordinary  gelatine  and 
in  sugar  gelatine  no  gas  is  pro- 
duced. 

Milk  :  Not  curdled  by  the  bacillus. 

Indol  :  The  production  of  indol  in  or- 
dinary broth  is  nil. 

Potato:  The  "invisible  growth,"  if 
potato  is  acid. 

Lactose  :  Fermentation  very  slight. 

25  per  cent.  Gelatine  at  37°  C. :  Strong- 
ly and  uniformly  turbid  (Klein). 

Eisner's  Iodised  Potato  Gelatine  :  Slow 
growth ;  small,  very  transparent 
colonies. 

Widal's  Test:  Bacilli  become  motion- 
less and  clumped  together  when 
suspended  in  a  drop  of  blood 
serum  from  a  typhoid  patient. 


B.  COLI 


Shorter,  thicker  ;  filaments  rare. 


Shorter,  stiffer,  fewer  ;  movement  less 
active. 

Even  edge,  homogeneous  ;  much 
larger,  quicker  growth,  and  less 
translucent  than;  B.  typhosus  ; 
medium  becomes  turbid  or  col- 
oured. 

Under  the  same  circumstances  abun- 
dant gas  is  produced. 

Milk  is  coagulated  (within  three  days). 
Indol  is  present. 

Thick,  yellow  growth. 

Fermentation  marked. 

Gelatine  remains  limpid  and  clear,  but 
possesses  thick  pellicle. 

Very  fast  growth  ;  larger,  brown,  less 
transparent  colonies. 

Bacilli  remain  actively  motile. 


BACTERIA    IN    WATER  59 

B.    TYPHOSUS  B.    COLI 

Broth  containing  Q.T)  per  cent.  Phenol  Grows  well. 
or  Formalin  (i :  7000):  No  growth. 

Thermal  Death  Point :  62°  C.  for  five  66°  C.  for  five  minutes  (Klein), 
minutes  (Klein). 

Vitality     in     Water    and     Sewage  :  The  B.  coli  retains  for  a  much  longer 
Typhoid  bacillus  soon  ceases  to  time  its  vitality  and  power  of  self- 

multiply  and  readily  dies  (Klein).  multiplication  (Klein). 

The  two  species,  Bacillus  typhosus  and  B.  coli,  agree  in 
possessing  the  following  characters :  no  spores,  no  liquefac- 
tion of  gelatine ;  both  grow  well  on  phenolated  gelatine,  and 
in  Parietti's  broth  ;  both  act  similarly  upon  animals,  though 
typhoid  fever  is  not  a  specific  disease  of  animals. 

The  Bacillus  typhosus,  though  a  somewhat  susceptible 
bacillus,  can  when  dried  retain  its  vitality  for  weeks.  In 
sewage  it  is  very  difficult  indeed  to  detect,  and  is  soon 
crowded  out.  Dr.  Andrews  and  Mr.  Parry  Laws,  in  their 
bacterial  researches  into  sewage  for  the  London  County 
Council,1  found  that  when  they  examined  specially  infected 
typhoid  sewage  it  was  only  with  extreme  difficulty  they 
isolated  Eberth's  bacillus.  In  ordinary  sewage  it  is  clear 
such  difficulty  would  be  greatly  enhanced. 

We  have  pointed  out  elsewhere  the  relation  between  soil 
and  typhoid.  In  water,  even  though  we  know  it  is  a  vehicle 
of  the  disease,  the  Bacillus  typhosus  has  been  only  very  rarely 
detected.  The  difficulties  in  separating  the  bacillus  from 
waters  (like  that  at  Maidstone,  for  example),  which  appear 
definitely  to  have  been  the  vehicle  of  the  disease,  are  mani- 
fold. To  begin  with,  the  enormous  dilution  must  be  borne 
in  mind,  a  comparatively  small  amount  of  contamination 
being  introduced  into  large  quantities  of  water.  Secondly, 
the  huge  group  of  the  B.  coli  species  considerably  compli- 
cates the  issues,  for  it  copiously  accompanies  the  typhoid, 
and  is  always  able  to  outgrow  it.  Further,  we  must  bear  in 

Report  on  the  Micro-organisms  of  Sewage,  Reports  to  L.  C.  C.,  1894, 
No.  216. 


60  BACTERIA 

mind  a  point  that  is  systematically  neglected,  namely,  that 
the  bacteriological  examination  of  a  water  which  is  suspected 
of  having  conveyed  the  disease  is  from  a  variety  of  circum- 
stances conducted  too  late  to  detect  the  causal  bacteria.  The 
incubation  period  of  typhoid  we  may  take  at  fourteen  days. 
Let  us  suppose  a  town  wrater  supply  is  polluted  with  some 
typhoid  excreta  on  the  1st  of  January.  Until  the  I4th  of 
January  there  may  be  no  knowledge  whatever  of  the  state 
of  affairs.  Two  or  three  days  are  required  for  notification  of 
cases.  Several  more  days  elapse  generally  before  bacterio- 
logical evidence  is  demanded.  Hence  arises  the  anomalous 


B.  COLI    COMMUNIS 

position  of  the  bacteriologist  who  sets  to  work  to  examine  a 
water  suspected  of  typhoid  pollution  three  weeks  previously. 
There  can  be  no  doubt  that  these  difficulties  are  very  real 
ones.  The  solution  to  the  problem  will  be  found  in  Dr. 
Klein's  dictum  that  "  a  water  in  which  sewage  organisms 
have  been  detected  in  large  numbers  should  be  regarded 
with  suspicion  "  '  as  the  vehicle  of  typhoid,  even  though  no 
typhoid  bacilli  were  discoverable.  The  chief  of  these  sewage 
bacteria  are  believed  to  be  Proteus  vulgaris,  B.  coli,  P.  zenkeri, 
and  B.  enteritidis,  and  they  are  all  nearly  related  to  B.  typhosus. 
The  presence  of  the  B.  coli  in  limited  numbers  is  not  suffi- 

1  Harben  Lectures,  1896. 


BACTERIA    IN    WATER  6 1 

cient  to  indicate  sewage  pollution,  seeing  that  it  is  so  widely 
distributed.  But  in  large  numbers,  and  in  company  with  the 
other  named  species,  it  is  almost  certain  evidence  of  sewage- 
polluted  water. 

It  may  occur  to  the  general  reader  that,  as  the  typhoid 
bacillus  is  not  extremely  rare,  drinking  water  may  frequently 
act  as  a  vehicle  to  carry  the  disease  to  man.  But,  to 
appreciate  the  position,  it  is  desirable  to  bear  in  mind  the 
following  facts :  the  typhoid  bacillus  is  only  found  in  the 
human  excrement  of  patients  suffering  from  the  disease  ; 
it  is  short-lived  ;  in  ordinary  waters  there  exist  organisms 
which  can  exert  an  influence  in  diminishing  its  vitality  ; 
exposure  to  direct  sunlight  destroys  it  ;  and  it  has  a  ten- 
dency to  be  carried  down-stream,  or  in  still  waters  settle 
at  the  bottom  by  subsidence.  Even  when  all  the  conditions 
are  fulfilled,  it  must  not  be  forgotten  that  a  certain  definite 
dose  of  the  bacillus  is  required  to  be  taken,  and  that  by 
a  susceptible  person.  Into  these  latter  questions  of  how 
bacteria  produce  disease  we  shall  have  an  opportunity  of 
inquiring  at  a  later  stage. 

We  must  now  mention  several  of  the  special  media  and 
tests  used  in  the  separation  of  Bacillus  typhosus  and  B.  coli. 

i.  The  Indol  Reaction.  Indol  and  skatol  are  amongst  the 
final  products  of  digestion  in  the  lower  intestine.  They  are 
formed  by  the  growth,  or  fermentation  set  up  by  the  growth, 
of  certain  organisms.  Indol  may  be  recognised  on  account 
of  the  fact  that  with  nitrous  acid  it  produces  a  dull  red 
colour.  The  method  of  testing  is  as  follows.  The  suspected 
organism  is  grown  in  pure  culture  in  broth,  and  incubated  for 
forty-eight  hours  at  37°  C.  Two  cc.  of  a  4  per  cent,  solution 
of  potassium  nitrite  are  added  to  100  cc.  of  distilled  water, 
and  about  I  cc.  of  this  is  added  to  the  test-tube  of  broth 
culture.  Now  a  few  drops  of  concentrated  sulphuric  acid 
(unless  quite  pure,  hydrochloric  should  be  used)  are  run 
down  the  side  of  the  tube.  A  pale  pink  to  dull  red  colour 


62  BACTERIA 

appears  almost  at  once,  and  may  be  accentuated  by  placing 
the  culture  in  the  blood-heat  incubator  for  half  an  hour. 
Much  dextrose  (derived  from  the  meat  of  the  broth)  inhibits 
the  reaction.  Bacillus  typhosus  does  not  produce  indol,  and 
therefore  does  not  react  to  the  test ;  B.  colt  and  the  bacillus 
of  Asiatic  cholera  do  produce  indol,  and  react  accordingly. 
It  should  be  pointed  out,  however,  that  the  bacillus  of  cholera 
also  produces  nitrites.  Hence  the  addition  of  acidonly  to  a 
peptone  culture  of  cholera  yields  the  "  red  reaction"  of  indol. 

2.  Carbolised  Gelatine.     To  ordinary  gelatine  .05  per  cent, 
of   phenol  is  added.       This  inhibits    many  common  water 
bacteria. 

3.  "  Shake  Cultures."     To  10  cc.  of  melted  gelatine  a  small 
quantity  of  the  suspected  organism  is  added.     The  test-tube 
is  then  shaken  and  incubated  at  22°  C.     If  the  organism  is 
Bacillus  coliy  the  next  day  reveals  a  large  number  of  gas- 
bubbles. 

4.  Eisner's  Medium.    This  special  potassium-iodide-potato- 
gelatine    medium  is  used    for  the  examination  of   typhoid 
excreta.       It    is   made   as    follows :     500   grams    of    potato 
gratings  are  added  to  1000  cc.  of  water ;  stand  in  cool  place 
for  twelve  hours,  and  filter  through  muslin  ;  add  150  grams 
of   gelatine ;   sterilise  and  add   enough  deci-normal  caustic 
soda  until  only  faintly  acid  ;  add  white  of  egg  ;  sterilise  and 
filter.    Before  use  add  half  a  gram  of  potassium  iodide  to 
every  50  cc.     Upon  this  acid  medium  common  water  bacteria 
will  not  grow,  but  Bacillus  typhosus  and  B.  coli  flourish. 

5.  Parietti's  Formula   consists   of — phenol,    five   grams; 
hydrochloric  acid,  four  grams  ;  distilled  water,  100  cc.       To 
10  cc.  of  broth  0.1-0.3  cc.  of  this  solution  is  added.     The 
tube  is  then  incubated  in  order  to  see  if  it  is  sterile.      If 
that  is  so,  a  few  drops  of  the  suspected  water  are  added, 
and  the  tube  reincubated  at  37°  C.  for  twenty-four  hours. 
If  the  water  contains  the  B.  typhosus  or  B.  coli,  the  tube  will 
show  a  turbid  growth. 


BACTERIA   IN   WATER  63 

6.  Widal  's  Reaction.    Mix  a  loopf  ul  of  blood  from  a  patient 
suspected  of  typhoid  fever  with  a  loopful  of  young  typhoid 
broth  culture  in  a  hanging  drop  on  a  hollow  ground  slide. 
Cover  with  a  cover  glass  and  examine  under  ^-inch  objective. 
If  the  patient  is  really  suffering  from  typhoid,  there  will  ap- 
pear in  the  hanging  drop  two  marked  characteristics,  viz., 
agglutination  and  immotility.      This  aggregation,  together 
with  loss  of  motility,  is  believed  to  be  due  to  the  inhibitory 
action  of  certain  bacillary  products  in  the  blood  of  patients 
suffering  from  the  disease.   The  test  may  be  applied  in  various 
ways,  and  its  successful  issue  depends  upon  one  or  two  small 
points  in  technique  into  which  we  cannot  enter  here,  but 
which  the  reader  will  find  dealt  with  in  the  appendix. 

7.  Flagella-staining.      Special  methods  must  be  adopted 
for  staining  the  flagella  of  Bacillus  typhosus  and  B.  coli.     The 
cover  glasses  should  be  absolutely  clean,  the  cultures  young 
(say  eighteen  hours  old),  and  a  diluted  emulsion  with  dis- 
tilled water  must  be  made  in  a  watch-glass  in  order  to  get 
bacilli    discrete   and    isolated    enough.      Van    Ermengems 
Method  is   as   follows : — Place   a   loopful   of   the   emulsion 
on  a  clean  cover  glass  and  dry  it  in  the  air,  fixing  it  lastly 
by  passing  it  once  or  twice  through  the  flame  of  a  Bunsen 
burner.     Place  films  for  thirty  minutes  in  a  solution  of  one 
part  boric  acid  (2  per  cent.)  and  two  parts  of  tannin  (15.25 
per  cent.),  which  also  contains  four  or  five  drops  of  glacial 
acetic   acid   to   every    100   cc.   of   the   mixture.     Wash   in 
distilled  water  and  alcohol.    Then  place  for  five  to  ten  seconds 
in  a  25.5  per  cent,  solution  of  silver  nitrate.     Immediately 
thereafter,  and  without  washing,  treat  the  cover  glass  to  the 
following  solution  for  two  or  three  seconds  :  gallic  acid,  five 
grams  ;  tannin,  three  grams ;    fused  potassium  acetate,  ten 
grams  ;  distilled  water,  350  cc.     After  this  place  in  a  fresh 
capsule  of  silver  nitrate  until  the  film  begins  to  turn  black. 
Wash  in  distilled  water,  dry,  and  mount.      The  process  con- 
tracts the  bacilli  somewhat,  but  the  flagella  stain  well. 


64  BACTERIA 

The  Bacillus  coli  communis  occupies  such  an  important 
place  in  all  bacteriological  investigation  that  a  few  words 
descriptive  of  it  are  necessary  in  this  place.  The  "  colon 
bacillus,"  as  it  is  termed,  appears  to  be  almost  ubiquitous  in 
distribution.  The  idea  once  held  that  it  belonged  exclusively 
to  the  alimentary  canal  or  sewage  is  now  discarded.  It  is 
one  of  the  most  widely  distributed  organisms  in  nature, 
though,  as  its  name  implies,  its  habitat  is  in  the  intestinal 
tract  of  man  and  animals.  It  is  an  aerobic,  non-sporulating, 
non-liquefying  bacillus,  about  .4  /*  in  thickness,  and  twice 
that  measurement  in  length ;  hence  it  often  appears  oval  or 
egg-shaped.  Its  motility  is  in  varying  degree,  occasionally 
being  as  active  as  B.  typhosus,  but  generally  much  less  so. 
It  possesses  lateral  flagella.  On  gelatine  plates  at  20°  C. 
B.  coli  produces  non-liquefying,  greyish-white,  round  colonies; 
in  a  stroke  culture  on  the  same  medium,  a  luxuriant  greyish 
band,  much  broader  and  less  restricted  to  the  track  of  the 
needle  than  B.  typhosus.  In  depth  of  medium  or  "  shake  " 
cultures  there  is  an  abundant  formation  of  bubbles  of  gas 
(methane  or  carbon  dioxide)  in  the  medium.  On  potato  it 
produces  a  light  yellow,  greasy  growth,  which  must  be  dis- 
tinguished from  the  growth  of  B.  fluorescens  liquefaciens, 
B.  pyocyaneus,  and  several  other  species  on  the  same  medium. 
If  the  potato  is  old  or  alkaline,  the  yellow  colour  may  not 
appear.  Milk  is  curdled  solid  in  from  twenty-four  to  forty- 
eight  hours,  and  a  large  amount  of  lactic  acid  produced. 
In  broth  it  produces  a  uniform  turbidity,  with  later  on 
some  sediment  and  a  slight  pellicle.  It  gives  the  reaction 
to  indol. 

It  is  now  the  practice  to  speak  of  the  family  of  Bacillus  coli 
rather  than  the  individual.  The  family  is  a  very  large  one, 
and  shows  throughout  but  few  common  characters.  The 
morphology  readily  changes  in  response  to  medium,  tem- 
perature, age,  etc.  Fermentation  of  sugar,  coagulation  of 
milk,  or  indeed  the  indol  reaction  cannot  always  be  used 


BACTERIA    IN    WATER  65 

as  final  tests  as  to  whether  or  not  the  organism  is  B.  coli,  for 
unfortunately  some  members  of  the  family  do  not  show  each 
of  these  three  features.  Most  varieties,  however,  appear 
to  show  some  motility,  a  small  number  of  flagella,  a  typical 
growth  on  potato,  and  develop  more  rapidly  on  all  media 
than  B.  typhosus.  These  characters,  plus  one  or  more  of 
the  three  features  above  named,  are  diagnostic  data  upon 
which  reliance  may  be  placed. 

Cholera.  This  word  is  used  to  cover  more  a  group  of  dis- 
eases rather  than  one  specific  well-restricted  disease.  In  recent 
years  it  has  become  customary  to  speak  of  Asiatic  cholera 
and  British  cholera,  as  if  indeed  they  were  two  quite  different 
diseases.  But,  as  a  matter  of  fact,  we  know  too  little  as  yet 
concerning  either  form  to  dogmatise  on  the  matter.  Until 
1884  practically  nothing  was  known  about  the  etiology  of 
cholera.  In  that  year,  however,  Koch  greatly  added  to  our 
knowledge  by  isolating  a  spirillum  from  the  intestine  and  in 
the  dejecta  of  persons  suffering  from  the  disease. 

Cholera  has  its  home  in  the  delta  of  the  Ganges.  From 
this  endemic  area  it  spreads  in  epidemics  to  various  parts  of 
the  world,  often  following  lines  of  communication.  It  is  a 
disease  which  is  characterised  by  acute  intestinal  irritation, 
manifesting  itself  by  profuse  diarrhoea  and  general  systemic 
collapse,  with  cramps,  cardiac  depression,  and  subnormal 
temperature.  The  incubation  period  varies  from  only  a  few 
hours  to  several  days.  In  the  intestine,  and  setting  up  its 
pathological  condition,  are  the  specific  bacteria ;  in  the 
general  circulation  their  toxic  products,  bringing  about  the 
systemic  changes.  Cholera  is  generally  conveyed  by  means 
of  water. 

The  spirillum  of  Asiatic  cholera  (Koch,  1884)  generally 
appears,  in  the  body  and  in  artificial  culture,  broken  into 
elements  known  as  "  commas."  These  are  curved  rods  with 
round  ends,  showing  an  almost  equal  diameter  throughout, 
and  sometimes  united  in  pairs  or  even  a  chain  (spirillum). 


66  BACTERIA 

The  latter  rarely  occur  in  the  intestine,  but  may  be  seen  in 
fluid  cultures.  The  common  site  for  Koch's  comma  is  in  the 
intestinal  wall,  crowding  the  lumina  of  the  intestinal  glands, 
situated  between  the  epithelium  and  the  basement  membrane, 


THE  COMMA-SHAPED  BACILLI  OF  CHOLERA 

abundant  in  the  detached  flakes  of  mucous  membrane,  and 
free  in  the  contents  of  the  intestine.  They  do  not  occur  in 
the  blood,  nor  are  they  distributed  in  the  organs  of  the 
body. 

The  bacilli  are  actively  motile,  and  possess  at  least  one 
terminal  flagellum.  The  organism  is  aerobic,  and  liquefies 
gelatine.  It  stains  readily  with  the  ordinary  aniline  dyes. 
It  does  not  produce  spores,  though  certain  refractile  bodies 
inside  the  protoplasm  of  the  bacillus  in  old  cultures  have 
been  regarded  as  such.  The  virulence  of  the  bacillus  is 
readily  attenuated,  and  both  the  virulence  and  morphology 
appear  to  show  in  different  localities  and  under  different 
conditions  of  artificial  cultivation  a  large  variety  of  what 
are  termed  involution  forms.  Unless  the  organism  is  con- 
stantly being  sub-cultured,  it  will  die.  Acid,  even  the  .2  per 
cent,  present  in  the  gastric  juice,  readily  kills  it.  Desiccation, 
55°  C.  for  ten  minutes,  and  weak  chemicals  have  the  same 
effect.  The  bacilli,  however,  have  comparatively  high  powers 
of  resistance  to  cold.  Unless  examined  by  the  microscope 


BACILLUS  TYPHOSUS  (SHOWING  FLAGELLA)      BACILLUS  TYPHOSUS  (WIDAL  REACTION) 

X  1000  (Agglutination  by  serum  from  typhoid  patient) 

X  400 


BACILLUS  COLI  COMMUNIS 

(From  agar  culture,  48  hours  growth) 
X  1000 


.«  IV 


- 


BACILLUS  MYCOIDES 

(Spore  formation.     P'rom  agar  culture) 
X  1000 


•By  permission  of  the  Scientific  Press,  Limited 


BACTERIA   IN    WATER  6/ 

in  a  fresh  and  young  stage,  it  is  difficult  to  differentiate 
Koch's  comma  from  many  other  curved  bacilli. 

Its  cultivation  characters  are  not  always  distinctive. 
Microscopically  the  young  colonies  in  gelatine  appear  as 
cream-coloured,  irregularly  round,  and  granular.  Lique- 
faction sets  in  on  the  second  day,  producing  a  somewhat 
marked  "  pitting  "  of  the  medium,  which  soon  becomes 
reduced  to  fluid.  In  the  depth  of  gelatine  the  growth  is 
very  characteristic.  An  abundant,  white,  thick  growth 
exactly  follows  the  track  of  the  needle,  here  and  there  often 
showing  a  break  in  continuity.  Liquefaction  sets  in  on  the 
second  day,  and  produces  a  distinctive  "  bubble  "  at  the 
surface.  The  liquefied  gelatine  does  not  fall  from  the  sides 
of  the  tube,  as  in  the  Finkler-Prior  comma  of  cholera  nostras, 
but  occurs  inside  the  border  where  the  gelatine  joins  the 
glass.  In  the  course  of  a  week  or  two  all  the  gelatine  may 
be  reduced  to  fluid.  On  agar  Koch's  comma  produces  with 
rapidity  a  thick,  greyish,  irregular  growth.  On  potato, 
especially  if  slightly  alkaline,  an  abundant  brownish  layer  is 
formed.  Broth  and  peptone  water  are  excellent  media. 
In  milk  it  rapidly  multiplies,  curdling  the  medium,  with 
production  of  acid.  Unlike  Bacillus  coli,  it  does  not  form 
gas,  but,  like  B.  coli,  it  produces  large  quantities  of  indol 
and  a  reduction  of  nitrates  to  nitrites.  Hence  the  indol  test 
may  be  applied  by  simply  adding  to  the  peptone  culture 
several  drops  of  strong  sulphuric  acid,  when  in  the  course 
of  several  hours,  if  not  at  once,  there  will  be  produced  a 
pink  colour,  the  "  cholera  red  reaction."  Although  it 
readily  loses  virulence,  and  its  resistance  is  little,  the  comma 
bacillus  retains  its  vitality  for  considerable  periods  in  moist 
cultures,  upon  moist  linen,  or  in  moist  soil.  In  cholera 
stools  kept  at  ordinary  room  temperature  the  cholera  bacil- 
lus will  soon  be  outgrown  by  the  putrefactive  bacteria.  The 
same  is  true  of  sewage  water. 

The  lower  animals  do  not  suffer  from  any  disease  at  all 


68  BACTERIA 

similar  to  Asiatic  cholera,  and  hence  it  is  impossible  to  fulfil 
the  postulate  of  Koch  dealing  with  animal  inoculation.  In 
this  respect  it  is  like  typhoid.  It  is,  however,  provisionally 
accepted  that  Koch's  bacillus  is  the  cause  of  the  disease. 
The  four  or  five  other  bacteria  which  have  from  time  to  time 
been  put  forward  as  the  cause  of  cholera  have  comparatively 
little  evidence  in  their  support.  It  is  less  from  these,  and 
more  from  several  spirilla  occurring  in  natural  waters,  that 
difficulties  of  diagnosis  arise. 

Some  hold  that,  however  many  comma  bacilli  be  intro- 
duced into  the  alimentary  canal,  they  will  not  produce  the 
disease  unless  there  is  some  injury  or  disease  of  the  wall  of 
the  intestine.  It  need  hardly  be  added  that  cholera  acts, 
like  other  pathogenic  bacteria,  by  the  production  of  toxins. 
Brieger  separated  cadaverin  and  putrescin  and  other  bodies 
from  cholera  cultures,  and  other  workers  have  separated  a 
tox-albumen. 

Methods  of  Diagnosis  of  Cholera  : 

1.  The  nature  of  the  evacuations  and  the  appearance  of 
the  mucous  membrane  of  the  intestine  afford  striking  evi- 
dence in  favour  of  a  positive  diagnosis.     Nevertheless  it  is 
upon  a  minute  examination  of  the  flakes  and  pieces  of  de- 
tached epithelium  that  reliance  must  be  placed.      In  these 
flakes  will  be  found  in  cholera  abundance  of  bacilli  having 
the  size,  shape,  and  distribution  of  the  specific  comma  of 
cholera.     The  size  and  shape  have  been  already  touched 
upon.      The    distribution    is    frequently    in    parallel    lines, 
giving  an  appearance  which  Koch  described  as  the  "  fish-in- 
stream  arrangement."     This  distribution  of  comma  bacilli 
in  the  flakes  of  watery  stools  is,  when  present,  so  character- 
istic of  Asiatic  cholera  that  it  alone  is  sufficient  for  a  definite 
diagnosis.     But  unfortunately  it  is  not  always  present,  and 
then  search  for  other  characters  must  be  made. 

2.  The  appearance  of  cultivation  on  gelatine,   to  which 
reference  has  been  made,  is  of  diagnostic  value. 


BACTERIA   IN    WATER  69 

3.  The  "  cholera  red  reaction."     It  is  necessary  that  the 
culture  be  pure  for  successful  reaction. 

4.  Isolation  from  water  is,  according  to  Dr.  Klein,  best 
accomplished  as  follows:  A  large  volume  of  water  (100-500 
cc.)  is  placed  in  a  sterile  flask,  and  to  it  is  added  so  much 
of  a  sterile  stock  fluid  containing  10  per  cent,  peptone,  5  per 
cent,  sodium  chloride,  as  will  make  the  total  water  in  the 
flask  contain  i  per  cent,  peptone  and  .5  per  cent.  salt.     Then 
the  flask  is  incubated  at  37°  C.      If  there  have  been  cholera 
vibrios  in  the  water,  however  few,  it  will  be  found  after 
twenty-four  hours'  incubation  that  the  top  layer  contains 
actively  motile  vibrios,  which  can  now  be  isolated  readily 
by  gelatine-plate  culture. 

5.  To  demonstrate  in   a  rapid   manner  the  presence  of 
cholera  bacilli  in  evacuations,  even  when  present  in  small 
numbers,  a  small  quantity  must  be  taken  up  by  means  of  a 
platinum  wire  and  placed  in  a  solution  containing  I  per  cent, 
of  pure  peptone  and  .  5  per  cent,  sodium  chloride  (Dunham). 
This  is  incubated  as  in  the  case  of  the  water,  and  in  twelve 
hours  is  filled  with  a  turbid  growth,  which  when  examined 
by  means  of  the  hanging  drop  shows  characteristic  bacilli. 

NATURAL    PURIFICATION   OF   WATER 

We  have  already  noticed  that  rivers  purify  themselves  as 
they  proceed.  There  are  many  excellent  examples  of  this 
self-purification.  The  Seine  as  it  runs  through  Paris  be- 
comes highly  polluted  with  every  sort  of  filthy  contamina- 
tion. But  twenty  or  thirty  miles  below  the  city  it  is  found 
to  be  even  purer  than  above  the  city  before  it  received  the 
city  sewage.  In  small  rivers  it  is  the  same,  provided  the 
pollution  is  less  in  amount.  Whilst  authorities  differ  with 
regard  to  the  mode  of  self-purification,  all  agree  that  in 
some  way  rivers  receiving  crude  sewage  are  able  in  a  mar- 
vellous degree  to  become  pure  again. 


7O  BACTERIA 

The  conditions  influencing  this  phenomenon  are  as  follows : 

(a)  The  Movement  of  the  Water.     It  is  probable,  however, 
that  any  beneficial  result  accruing  from  this  cause  is  due,  not 
to  any  mechanical  factor  in  the  movement,  but  to  the  extra 
surface  of  water  available  for  oxidation  processes. 

(b)  The  Pressure  of  the   Water.     It  is  believed  that  the 
volume  of  water  pressing  down  upon  any  given  area  be- 
neath it  weakens  the  vitality  of  certain  microbes.     In  sup- 
port of  this  theory,  it  is  urged  that  the  number  of  bacteria 
capable  of  developing  is  less  the  greater  the  depth  from  the 
surface.     Yet  it  must  be  remembered  that  mud  at  the  bot- 
tom of  a  river,  or  at  the  bottom  of  the  sea,  is  teeming  with 
living  organisms. 

(c)  Light.     We  have  seen  how  prejudicial  is  light  to  the 
growth  of  organisms  in  culture  media.     This  is  so,  though 
to  a  less  extent,  in  water.     Arloing  held  that  sunlight  could 
not  pierce  a  layer  of  water  an  inch  in  thickness  and  still  act 
inimically  on  micro-organisms.     But  Buchner  found  that  the 
sun's  rays  could  pass  through  fifteen  or  twenty  inches  and 
yet  be  bactericidal.     This  evidence  appears  contradictory. 
On  the  whole,  however,  authorities  agree  that  the  influence 
of  the  sun's  rays  upon  water  is  distinctly  bactericidal  and 
causes  a  marked  diminution  in  the  quantity  of  organisms 
after  acting  for  some  hours.    Especially  will  this  be  so  when 
the  water  is  spread  over  a  wide  area  and  is  therefore  shallow 
and  stationary,  or  moving  but  slowly. 

(d)  Vegetation  in  Water.     Pettenkofer,  in  his  observations 
upon  the  Iser  below  Munich,  has  shown  how  algae  bring 
about  a  marked  reduction  in  the  organic  matters  present  in 
water. 

(e)  Dilution.     There  can  be  no  doubt  in  anyone's  mind 
that  the  pollutions  passing  into  a  flowing  river  are  very  soon 
diluted    with   the   large   quantities   of  comparatively  pure 
water  always  forthcoming.     But  this,  whilst  it  would  lower 
the  percentage  of  impurity,  cannot  remove  impurities. 


BACTERIA   IN    WATER  J\ 

(/)  Sedimentation.  Whilst  Pettenkofer  attributes  self- 
purification  to  oxygenation  and  vegetation,  most  authorities 
are  now  agreed  that  it  is  largely  brought  about  by  the  sub- 
sidence of  impure  matters,  and  by  their  subsequent  dis- 
integration at  the  bottom  of  the  river.  Sedimentation 
obviously  is  greatest  in  still  waters.  Hence  lake  water 
contains  as  a  rule  very  few  bacteria.  '  The  improvement 
in  water  during  subsidence  is  the  more  rapid  and  pronounced 
the  greater  the  amount  of  suspended  matter  initially  pre- 
sent "  (Frankland).  Tils  has  pointed  out  that  the  number 
of  micro-organisms  was  invariably  smaller  in  the  water  col- 
lected from  the  reservoir  than  in  that  taken  from  the  source 
supplying  the  latter.  Percy  Frankland  has  demonstrated 
the  same  effect  of  sedimentation  by  storage  as  follows : 

No.  of  Colonies  in 
i  cc.  of  water. 

1.  Intake  from  Thames,  June  25,  1892 ^QQ1 

2.  First  small  storage  reservoir !,7O3 

3.  Second  "  "          1,156 

4.  Large  storage  reservoir 464 

The  large  reservoir  would  of  course  necessitate  a  prolonged 
subsidence,  and  hence  a  greater  diminution  than  in  the  small 
reservoirs.  Many  like  examples  might  be  cited,  but  a  typi- 
cal one  such  as  the  above  will  suffice. 

(g)  Oxidation.  Many  experiments  and  observations  have 
been  made  to  prove  that  large  quantities  of  oxygen  are  used 
up  daily  in  oxygenising  the  Thames  water.  Oxygenated 
water  will  come  up  with  the  tide  and  down  with  the  fresh 
water  from  above  London.  There  will  also  be  oxygen  ab- 
sorption going  on  upon  the  surface  of  the  water,  and  from 
these  three  sources  enough  oxygen  is  obtained  to  oxidise 
impurities  and  produce  what  is  really  an  effluent.  In  many 
smaller  streams  the  opportunity  for  oxidation  is  afforded  by 
weirs  and  falls. 

Probably  all  these  factors  play  a  part  in  the  self-purifica- 


72  BA  CTERIA 

tion  of  rivers,  but  we  may  take  it  that  oxidation,  dilution, 
and  sedimentation  are  three  of  the  principal  agencies. 

We  may  here  digress  to  refer  in  passing  to  the  facts  ob- 
tainable from  Sir  Edward  Frankland's  report  on  Metropoli- 
tan water  supply  in  1894,  as  they  will  afford  a  connecting  link 
between  self  purification  and  artificial  purification.  Judged 
by  the  relatively  low  proportion  of  carbon  to  nitrogen,  the 
organic  matter  present  in  the  water  was,  as  usual,  found  to 
be  chiefly,  if  not  entirely,  of  vegetable  origin.  An  immense 
destruction  of  bacteria  was  found  to  be  effected  by  storage 
in  subsidence  reservoirs.  The  bacterial  quality  of  the  water 
might  differ  widely  from  its  chemical  qualities.  These 
three  facts  are  of  primary  importance  in  the  interpretation 
of  water  reports,  and  it  will  be  well  to  bear  them  in  mind. 
Sir  E.  Frankland  also  refers  to  the  physical  conditions 
affecting  microbial  life  in  river  waters.  The  importance  of 
changes  of  temperature,  the  effect  of  sunlight,  and  rate  of 
flow  had  been  referred  to  in  previous  reports.  Respecting 
the  relative  proportion  of  these  factors,  he  adds : 

"  The  number  of  microbes  in  Thames  water  is  determined 
mainly  by  the  flow  of  the  river,  or,  in  other  words,  by  the  rain- 
fall, and  but  slightly,  if  at  all,  by  either  the  presence  or  absence  of 
sunshine,  or  a  high  or  low  temperature.  With  regard  to  the 
effect  of  sunshine,  the  interesting  researches  of  Dr.  Marshall 
Ward  leave  no  doubt  that  this  agent  is  a  powerful  germicide,  but 
it  is  probable  that  the  germicidal  effect  is  greatly  diminished,  if 
not  entirely  prevented,  when  the  solar  rays  have  to  pass  through 
a  comparatively  thin  stratum  of  water  before  they  reach  the  living 
organisms." 

From  which  it  is  clear  that  evidence  favours  the  effect  of 
sedimentation  and  dilution.  These  two  factors  in  conjunc- 
tion with  filtration  are,  practically  speaking,  the  methods  of 
artificial  water  purification,  with  which  we  are  now  in  a 
position  to  deal. 


BACTERIA   IN    WATER  73 

ARTIFICIAL  PURIFICATION   OF  WATER 

Sedimentation  and  Precipitation.  Naturally,  we  see  this 
factor  in  action  in  lakes  or  reservoirs.  For  example,  the 
water  supply  of  Glasgow  is  the  untreated  overflow  from 
Loch  Katrine.  Purification  has  been  brought  about  by 
means  of  subsidence  of  impurities.  Nothing  further  is 
needed.  Artificially,  we  find  it  is  this  factor  which  is 
the  mechancial  purifier  of  biological  impurity  in  such 
methods  as  Clark 's  process.  By  this  mode  "  temporary 
hardness,"  or  that  due  to  soluble  bicarbonate  of  lime,  is 
converted  into  insoluble  normal  carbonate  of  lime  by  the 
addition  of  a  suitable  quantity  of  lime-water.  Carbonates 
of  lime  and  magnesia  are  soluble  in  water  containing  free 
carbonic  acid,  but  when  fresh  lime  is  added  to  such  water 
it  combines  with  the  free  CO3  to  form  the  insoluble  carbon- 
ate, which  falls  as  a  sediment : 

CaCO3  +  CO2  +  CaH2O2  (lime-water)  =  2  CaCO3  +  H2O. 

As  the  carbonate  falls  to  the  bottom  of  the  tank  it  carries 
down  with  it  the  organic  particles.  Hence  sedimentation 
is  brought  about  by  means  of  chemical  precipitation.  It  is 
obviously  a  mechanical  process  as  regards  its  action  upon 
bacteria.  Nevertheless  its  action  is  well-nigh  perfect,  and 
300  or  400  m.-o.  per  cc.  are  reduced  to  4  or  5  per  cc.  We 
shall  refer  to  this  same  action  when  we  come  to  speak  of 
bacterial  purification  of  sewage.  Alum  has  been  frequently 
used  to  purify  waters  which  contain  much  suspended  mat- 
ter. Five  or  six  grains  of  alum  are  added  to  each  gallon  of 
water,  with  some  calcium  carbonate  by  preference.  Precipi- 
tation occurs,  and  with  it  sedimentation  of  the  bacteria,  as 
before.  But,  as  Babes  has  pointed  out,  alum  itself  acts 
inimically  on  germs;  in  such  treatment,  therefore,  we  get 
sedimentation  and  germicidal  action  combined. 

As  a  matter  of  actual  practice,  however,  sedimentation 


74  BACTERIA 

alone  is  rarely  sufficient  to  purify  water.  It  is  true  that  the 
collection  of  water  in  large  reservoirs  permits  subsidence  of 
suspended  matters,  and  affords  time  for  the  action  of  light 
and  the  competitive  suicidal  behaviour  of  the  common  water 
bacteria.  Yet,  after  all,  filtration  is  the  most  important 
and  most  reliable  method. 

Sand  Filtration,  as  a  means  of  purifying  water,  has  been 
practised  since  the  early  part  of  the  present  century.  But 
it  was  not  till  1885  that  Percy  Frankland  first  demonstrated 
the  great  difference  in  bacterial  content  between  a  water  un- 
filtered  and  a  water  which  had  passed  through  a  sand  filter. 
Previous  to  this  time  the  criterion  of  efficiency  in  water  puri- 
fication had  been  a  chemical  one  only,  and  the  presence  or 
absence  of  bacteria  in  any  appreciable  quantity  was  described, 
not  in  mathematical  terms,  but  in  indefinite  descriptive 
words,  like  "  turbid,"  "cloudy,"  etc.  It  is  needless  to  say 
that  this  difference  in  estimation  was  due  to  the  introduction 
by  Koch  of  the  gelatine-plate  method  of  examination.  As 
a  result  of  Percy  Frankland's  work,  he  formulated  the  fol- 
lowing conclusions  as  regards  the  chief  factors  influencing 
the  number  of  microbes  passing  through  the  filter. 

It  depends  upon: 

(1)  The  Storage  Capacity  for  Unfiltered  Water.     This,  of 
course,  has  reference    to   the    advantages,  which  we  have 
noticed  above,  of  securing  a  large  collection  of  water  pre- 
vious to  filtration  for  subsidence,  etc. 

(2)  The  Thickness  of  Fine  Sand  through  which  Filtration  is 
Carried  on.     An  argument  needing  no  further  support,  for 
it  is  clear,  other  things  being  equal,  the  more  sand  water 
passes  through  the  greater  the  opportunity  of  leaving  its 
impurities  behind. 

(3)  Rate  of  Filtration.     The  slower  filtration  will  be  gen- 
erally the  more  complete  in  its  results. 

(4)  Renewal  of  Filter-Beds.     After  a  certain  time  the  filter- 
bed  becomes  worn  out  and  inefficient ;  at  such  times  renewal 


BACTERIA   IN    WATER  75 

is  necessary.  Not  only  may  the  age  of  the  filter  act  preju- 
dicially, but  the  extra  pressure  required  will  tend  to  force 
through  it  bacteria  which  ought  to  have  remained  in  the  filter. 
In  1893,  Koch  brought  out  his  monograph  upon  Water 
Filtration  and  Cholera,  and  his  work  had  a  deservedly  great 
influence  upon  the  whole  question.  He  shows  how  the 
careful  filtration  of  water  supplied  to  Altona  from  the  Elbe 
saved  the  town  from  the  epidemic  of  cholera  which  came 
upon  Hamburg  as  a  result  of  drinking  unfiltered  water,  al- 
though Altona  is  situated  several  miles  below  Hamburg,  and 
its  drinking  water  is  taken  from  the  river  after  it  has  received 
the  sewage  of  Hamburg.  Now,  from  his  experience  of 
water  filtration,  Koch  arrived  at  several  important  conclu- 
sions. In  the  first  place,  he  maintained  that  the  portion  of 
the  filter-bed  which  really  removed  micro-organisms  effectively 
was  the  slimy  organic  layer  upon  the  surface.  This  layer  is 
produced  by  a  deposit  from  the  still  unpurified  water  lying 
immediately  above  it.  The  most  vital  part  of  the  filter-bed 
is  this  organic  layer,  which,  after  formation,  should  not  be 
disturbed  until  it  requires  removal  owing  to  its  imperme- 
ability. A  filter-bed,  as  is  well  known,  consists  of  say  three 
feet  of  sand  and  one  foot  of  coarse  gravel.  The  water  to 
be  filtered  is  collected  into  large  reservoirs,  where  subsi- 
dence by  gravitation  occurs.  Thence  it  is  led  by  suitable 
channels  to  the  surface  of  the  filter-bed.  Having  passed 
through  the  three  or  four  feet  of  the  bed,  it  is  collected  in  a 
storage  reservoir  and  awaits  distribution.  The  action  of  the 
whole  process  is  both  mechanical  and  chemical.  Mechanic- 
ally by  subsidence,  much  suspended  matter  is  left  behind  in 
the  reservoir.  Again,  mechanically,  much  of  that  which 
remained  suspended  in  the  water  when  it  reached  the  filter- 
bed  is  waylaid  in  the  substance  of  the  sand  and  gravel  of  the 
filter-bed.  Chemically  also  the  action  is  twofold.  Oxidation 
of  the  organic  matter  occurs  to  some  extent  as  the  water 
passes  through  the  sand.  Until  recently  this  chemical  action 


76  BA  CTERIA 

and  the  double  mechanical  action  were  believed  to  be  the  com- 
plete process,  and  its  efficiency  was  tested  by  chemical  oxida- 
tion and  alteration,  and  absence  of  the  suspended  matter. 

Now,  however,  it  is  recognised  that  the  second  portion 
of  the  chemical  action  is  vastly  the  more  important,  indeed, 
the  only  vital,  part  of  the  process.  This  is  the  chemical 
effect  of  the  layer  of  scum  and  mud  on  the  surface  of  the 
sand  at  the  top  of  the  filter-bed.  The  mechanical  part  of 
this  layer  is,  of  course,  the  holding  back  of  the  particulate 
matter  which  has  not  subsided  in  the  reservoir;  the  vital 
action  consists  in  what  is  termed  nitrification  of  unoxidised 
substance,  which  is  accomplished  in  this  layer  of  organic 
matter.  We  shall  deal  at  some  length  with  the  principles 
of  nitrification  when  we  come  to  speak  of  soil.  But  we  may 
say  here  that  by  nitrification  is  understood  a  process  of 
oxidation  of  elementary  compounds  of  nitrogen,  by  which 
these  latter  are  built  up  into  stable  bodies  which  can  do 
little  harm  in  drinking  water.  From  what  has  been  said  it 
will  be  seen  that  the  action  of  a  filter-bed  is  of  a  complicated 
nature.  There  is  (i)  subsidence  of  the  grosser  particles  of 
impurity  in  the  water;  (2)  mechanical  obstruction  to  impuri- 
ties in  the  interstices  of  the  scum,  sand,  and  gravel  in  the 
filter;  (3)  oxidation  of  organic  matter  by  the  oxygen  held  in 
the  pores  of  the  sand  and  gravel;  (4)  nitrification  in  the 
vital  scum  layer,  which  is  accomplished  by  micro-organisms 
themselves.  This  latter  is  now  considered  to  be  incompar- 
ably the  most  important  part  of  the  filter.  That  being  so, 
its  removal,  except  when  absolutely  necessary,  is  to  be 
avoided  as  detrimental  to  the  efficiency  of  the  filter.  New 
filters  have  obviously  but  little  of  this  action.  Hence  it  is 
wise  to  allow  a  new  filter-bed  to  act  for  a  short  period  (say 
twenty-four  to  forty-eight  hours)  before  the  filtered  water  is 
used  for  domestic  purposes,  in  order  to  allow  the  organic 
layer  to  be  formed.  This  must  also  be  borne  in  mind  after 
a  filter-bed  has  been  cleaned. 


BACTERIA   IN    WATER  77 

To  maintain  this  nitrifying  action  of  a  filter  in  efficiency, 
Koch  suggested,  in  the  second  place,  that  the  rate  of  filtra- 
tion must  not  exceed  four  inches  per  hour.  At  the  Altona 
water-works  this  rate  of  filtration  was  maintained,  and  the 
number  of  organisms  always  remained  below  100  per  cc., 
which,  as  we  have  seen,  is  the  standard.  Thirdly,  it  is  im- 
portant that  periodic  bacteriological  examinations  should  be 
made.  Koch's  emphasis  upon  this  point  is  well  known,  and 
the  cumulative  experience  of  bacteriologists  only  further 
supports  such  a  course  being  taken.  If  it  be  true  that 
efficient  sand  filtration  is  a  safeguard  against  pathogenic 
germs  like  typhoid  and  cholera,  then  there  can  be  but  one 
criterion  of  efficiency,  viz.,  their  absence  in  the  filtered 
water,  which  can  only  be  ascertained  by  regular  examina- 
tion. But  it  is  not  alone  for  pathogenic  germs  that  filtration 
is  proposed.  Filtered  water  containing  more  than  100 
micro-organisms  of  any  kind  per  cc.  is  below  the  standard 
in  purity,  and  should  on  no  account  be  distributed  for  drink- 
ing purposes.  In  this  country  chemical  analysis,  with  a 
more  or  less  cursory  microscopic  examination,  has  been 
almost  invariably  accepted  as  reliable  indication  of  the  con- 
dition of  the  water.  But  such  an  examination  is  not  really 
any  more  a  fair  test  of  the  working  of  the  filter  than  it  is  of 
the  actual  condition  of  the  water.  It  is  true,  the  quantity 
of  organic  matter  can  be  estimated  and  the  condition  in 
which  it  exists  in  combination  obtained ;  but  it  cannot  tell 
us  what  a  bacteriological  examination  can  tell  us,  viz.,  the 
quantity  and  quality  of  living  micro-organisms  present  in 
the  water.  Upon  this  fact,  after  all,  an  accurate  conclusion 
depends.  There  is  abundant  evidence  to  show  that  no 
valuable  opinion  can  be  passed  upon  a  water  except  by  both 
a  chemical  and  a  bacteriological  examination,  and  further 
by  a  personal  investigation,  outside  the  laboratory,  of  the 
origin  of  the  water  and  its  liabilities  to  pollution.' 

So  convinced  was  Koch  of  the  efficiency  of  sand  filtration 


BACTERIA 


as  protection  against  disease-producing  germs  that  he  advo- 
cated an  adaptation  of  this  plan  in  places  where  it  was  found 
that  a  well  yielded  infected  water.  Such  pollution  in  a  well 
may  be  due  to  various  causes ;  surface-polluted  water  oozing 
into  the  well  is  probably  the  commonest,  but  decaying 
animal  or  vegetable  matter  might  also  raise  the  number  of 
micro-organisms  present  almost  indefinitely.  Koch's  pro- 
posal for  such  a  polluted  well  was  to  fill  it  up  with  gravel  to 
its  highest  water  level,  and  above  that,  up  to  the  surface  of 
the  ground,  with  fine  sand.  Before  the  well  is  filled  up  in 
this  manner  it  must,  of  course,  be  fitted  with  a  pipe  passing 
to  the  bottom  and  connected  with  a  pump.  This  simple 
procedure  of  filling  up  a  well  with  gravel  and  sand  interposes 
an  effectual  filter-bed  between  the  subsoil  water  and  any 
foul  surface  water  percolating  downwards.  Such  an  arrange- 
ment yields  as  good,  if  not  better,  results  than  an  ordinary 
filter-bed,  on  account  of  there  being  practically  no  disturb- 
ance of  the  bed  nor  injury  done  to  it  by  frost. 

The  effect  of  the  remedies  we  have  been  discussing  upon 
the  number  of  bacteria  is  demonstrated  in  the  results  which 
Sir  Edward  Frankland  arrived  at  in  his  investigation  of 
London  waters.1 

MEAN  OF  MONTHLY  EXAMINATIONS  FOR  THE  YEAR 


NAME  OF  COMPANY. 

Source  of 
Supply. 

M.-O.  PER  CC. 

Average  per 
cent,  of 
Micro-organ- 
isms Removed 
by  Filtration. 

At 
Source. 

After 
Storage. 

_  After 
Filtration. 

The  Chelsea  Co 

Thames  at 
Hampton 

16,138 
16,138 
16,138 

16,138 
16,138 

1067 

1788 

2500 
7820 

34 
58 
80 
(623 
•<  100 

(  96 

75 

98.96 
99.40 
97.72 

j-  98-46 
99-50 

West  Middlesex  Co  

Southwark  &  Vauxhall  Co. 
Grand  Junction  Co  

Lambeth  Co  

Report  on  the  Metropolitan  Water  Supply. 


BACTERIA   IN    WATER  79 

The  teaching  of  these  figures  could,  with  great  ease,  be 
reproduced  again  and  again  if  such  was  necessary ;  but  these 
will  suffice  to  show  that  sand  filtration,  when  carefully 
carried  out,  offers  a  more  or  less  absolute  barrier  to  the 
passage  of  bacteria,  whether  non-pathogenic  or  pathogenic. 

Domestic  Purification  of  Water.  Something  may  here  be 
said,  from  a  bacteriological  point  of  view,  relative  to  what 
is  called  domestic  purification.  There  is  but  one  perfectly 
reliable  method  of  sterilising  water  for  household  use,  viz., 
boiling.  As  we  have  seen,  moist  heat  at  the  boiling  point 
maintained  for  five  minutes  will  kill  all  bacteria  and  their 
spores.  The  only  disadvantages  to  this  process  are  the 
labour  entailed  and  the  "  flat  "  taste  of  the  water.  Never- 
theless in  epidemics  due  to  bad  water  it  is  desirable  to  revert 
to  this  simple  and  effectual  purification. 

There  are  a  large  number  of  filters  on  the  market  with,  in 
many  cases,  but  little  modification  from  each  other.  The 
materials  out  of  which  they  are  made  are  chiefly  the  follow- 
ing: carbon  and  charcoal,  iron  (spongy  iron  or  magnetic 
oxide),  asbestos,  porcelain  and  other  clays,  natural  porous 
stone,  and  compressed  siliceous  and  diatomaceous  earths. 
From  an  extended  research  in  1894  by  Dr.  Sims  Woodhead 
and  Dr.  Cartwright  Wood  our  knowledge  of  the  quality  of 
these  substances  as  protectives  against  bacteria  has  been 
largely  increased.  They  concluded  that  a  filter  failed  to  act 
in  one  of  two  ways.  It  was  either  pervious  to  micro-organ- 
isms, or  its  power  of  filtering  became  modified  owing  to  (a) 
structural  alteration  of  its  composition,  or  to  (b)  the  growing 
through  of  the  micro-organisms.  The  conditions  which 
chiefly  influence  the  growth  of  bacteria  through  a  filter  ap- 
pear to  be  the  temperature,  the  intermittent  use  of  the  filter, 
and  the  species  of  bacteria.  The  higher  the  temperature 
and  the  longer  the  organisms  are  retained  in  the  filter 
the  more  likely  is  it  that  they  will  grow  through,  and  in 
the  next  usage  of  the  filter  appear  in  the  filtrate.  As  to  the 


8o 


BACTERIA 


species,  those  multiplying  rapidly  and  possessing  the  power 
of  free  motility  will  naturally  appear  earlier  in  a  filtrate 
than  others.  Woodhead  and  Wood,  from  their  searching 
and  most  able  investigation,  concluded  that  the  Pasteur- 
Chamberland  candle  filters  (composed  of  por- 
celain formed  by  a  mixture  of  kaolin  and  other 
clays)  were  the  only  filters  out  of  the  substances 
named  above  which  were  reliable  and  protective 
against  bacteria.  They  tested  over  three  dozen 
of  the  Pasteur  filters,  and  "  in  every  case  these 
gave  a  sterile  filtrate."  Pure  cholera  bacillus 
in  suspension  (5000  bacilli  to  every  cc.)  and 
typhoid  bacillus  in  suspension  (8000  per  cc.) 
were  passed  through  these  filters,  and  not  a 
single  bacillus  was  detectable  in  the  filtrate. 
The  Berkefeld  filter  (siliceous  earth)  came 
second  on  the  list  as  an  effective  filter,  and  had 
but  the  fault  of  not  being  a  "  continuous  " 
steriliser.  A  certain  Parisian  filter  ("  Por- 
celaine  d'Amiante  "),  made  of  unglazed  porce- 
lain, rendered  water  absolutely  free  from 
bacteria.  Its  action  was,  however,  very  slow. 

Setting  aside  these  three  efficient  filters,  we 
are  face  to  face  with  the  fact  that  most  filters 
^o  no|-  produce  germ-free  filtrates,  even  though 
they  are  nominally  guaranteed  to  do  so.  It  is  professed 
for  animal  charcoal,  which  is  widely  used,  that  it  absorbs 
oxygen,  and  so  fully  oxidises  whatever  passes  through  it. 
This  may  be  so  at  first,  but  after  a  little  use  it  probably 
does  more  harm  than  good.  It  appears  to  add  nitrogen 
and  phosphates  to  water,  which  are  both  nutritive  sub- 
stances on  which  bacteria  grow.  Moreover  it  readily  ab- 
sorbs impurities  from  the  air.  As  a  matter  of  experiment 
and  practice,  it  has  been  found  by  Frankland,  Woodhead, 
and  others,  that  charcoal  actually  adds  to  the  number  of 
germs  after  it  has  been  in  use  for  some  days. 


|o 


PASTEUR- 


Attached  to 
water  Supply 


BACTERIA   IN    WATER  8 1 

Diseases  Conveyed  by  Water.  There  are  a  few  preliminary 
features  to  be  noticed  before  we  enter  in  detail  upon  the 
characteristics  of  several  of  the  chief  pathogenic  bacteria  in 
water. 

In  sterilised  water,  and  in  very  highly  polluted  water  or 
sewage,  pathogenic  bacteria  do  not  flourish.  In  the  former 
case  they  die  of  starvation,  although  there  are  some  experi- 
ments on  record  which  appear  not  to  support  this  view ;  in 
the  latter  case  they  are  killed  by  the  enormous  competition 
of  common  bacteria.  Even  in  ordinary  water  there  is  a 
wide  divergence  of  behaviour.  Some  bacteria  are  destroyed 
in  a  few  hours;  others  appear  to  flourish  for  weeks.  In  all 
cases  the  spores  are  able  to  resist  whatever  injurious  proper- 
ties the  water  may  have  much  more  persistently  than  the 
bacilli  themselves.  These  changes  in  the  vitality  of  bacteria 
in  water,  partly  due  to  the  water  and  partly  to  the  other 
micro-organisms,  bring  about  two  characteristics  which  it  is 
important  to  remember,  viz.,  that  pathogenic  germs  in  water 
are,  as  a  rule,  scanty  and  intermittent.  It  is  these  features 
in  conjunction  with  the  enormous  quantities  of  common 
water  bacteria  which  make  the  search  for  the  bacillus  of 
typhoid  what  Klein  has  called  "  searching  for  a  needle  in  a 
rick  of  hay."  Not  that  it  cannot  be  detected,  but  its  de- 
tection is  one  of  the  most  difficult  of  investigations.  We 
shall  refer  to  this  matter  again  when  Bacillus  typhosus  is 
under  consideration. 

In  artificial  cultivation  water  bacteria  respond  very  readily 
to  external  conditions.  Increase  of  alkalinity  (.01  grams  of 
sodium  carbonate  added  to  10  cc.  of  ordinary  gelatine) 
causes  the  number  of  colonies  to  be  five  or  six  times  greater 
than  that  revealed  by  using  ordinary  gelatine ;  on  the  other 
hand,  very  slightly  increasing  the  acidity  of  a  medium  as 
markedly  diminishes  the  number  of  bacteria.  Advantage  is 
taken  of  this  in  culturing  the  bacillus  of  typhoid,  which  does 
not  object  to  an  acid  medium. 

Water  may  become  polluted  in  a  variety  of  ways,  and  it 


82  BACTERIA 

is  helpful  to  classify  these  as  pollutions  at  the  source,  in  the 
course,  and  at  the  periphery.  Gathering-grounds  are  fre- 
quently the  locality  of  the  pollution.  The  recent  Maidstone 
epidemic  is  an  example.  Here  some  of  the  springs  supply- 
ing the  town  with  water  were  contaminated  by  several 
typhoid  patients.  Frequently  on  the  gathering-ground  one 
may  find  a  number  of  houses  the  waste  and  refuse  of  which 
will  furnish  ample  surface  pollution,  which  in  its  turn  may 
readily  pass  into  a  collecting  reservoir  or  a  well.  Only  re- 
cently the  writer  investigated  the  cause  of  typhoid  in  a  large 
country  house,  and  traced  it  to  pollution  of  the  private  well 
by  surface  washings  from  the  stable  quarters.  Leakage  of 
house-drains  into  wells  is  not  an  infrequent  source  of  con- 
tamination. The  same  cause  is  generally  operative  in  cases 
of  pollution  of  a  water  supply  in  its  course  from  the  source 
to  the  cisterns  or  taps  at  the  periphery,  viz.,  a  sewer  or  drain 
leaking  into  the  water  supply. 

Water  companies  and  those  responsible  for  water  supply 
appear  to  hold  the  opinion  that  so  long  as  there  is  sand 
filtration  or  subsidence  reservoirs  it  is  unnecessary  to  con- 
sider the  gathering-ground  or  transit.  But,  as  we  have 
seen,  a  frost  may  completely  dislocate  the  efficient  action 
of  a  filter,  and  times  of  flood  may  prevent  proper  sediment- 
ation ;  then  our  dependence  for  pure  water  is  wholly  upon 
the  gathering-ground  and  source.  Hence  we  find  water  con- 
taminated at  its  source  by  polluted  wells,  by  sewage-infected 
rivers  and  streams,  by  drainage  of  manured  fields,  by  in- 
numerable excremental  pollutions  over  the  areas  of  the 
gathering-grounds,  and  in  transit  by  careless  laying,  poor 
construction,  bad  jointing,  and  close  proximity  of  water-  and 
drain-pipes.  In  the  third  place,  we  may  get  a  water  infected 
at  the  periphery,  in  the  house  itself.  Such  cases  are  generally 
due  to  one  of  two  causes :  filthy  cisterns  or  suction.  Cisterns 
per  se  are  more  or  less  indispensable  where  a  constant  service 
does  not  exist,  but  they  should  be  inspected  from  time  to 


BACTERIA    IN    WATER  83 

time  and  maintained  in  a  cleanly  condition.  Suction  into 
the  tap  has  been  recently  emphasised  by  Dr.  Vivian  Poore 
as  a  cause  of  pollution.  It  is  liable  to  occur  whenever  a  tap 
is  left  turned  on,  and  a  vacuum  is  produced  in  the  supply- 
pipe  by  intermission  of  the  water  supply,  so  that  foul  gas  or 
liquid  is  sucked  back  into  the  house-pipe. 

One  more  point  requires  our  attention.  It  has  relation 
to  bacterially  polluted  water  when  it  has  gained  entrance  to 
the  body.  It  has  been  known  for  some  time  past  that  not 
all  waters  polluted  with  disease  germs  produce  disease.  As 
we  have  before  said,  this  may  depend  upon  the  infective 
agent,  its  quantity  and  quality;  the  body  being  able  in 
many  cases  to  resist  a  small  dose  of  poison.  It  is,  however, 
necessary  to  infection,  especially  in  water-borne  disease,  that 
the  tissues  shall  be  in  some  degree  disordered.  The  per- 
verted action  of  the  stomach  influences  the  acid  secretion 
of  the  gastric  juice,  through  which  bacilli  might  then  pass 
uninjured.  Particularly  must  this  be  so  in  the  bacillus  of 
cholera,  which  is  readily  killed  by  the  normal  acid  reaction 
of  the  stomach.  Hence,  in  this  disease  at  least,  it  is  the 
opinion  of  bacteriologists  that  the  condition  of  the  mucous 
membrane  of  the  stomach  is  of  primary  importance.  Metsch- 
nikoff  has  indeed  demonstrated  the  presence  of  the  bacil- 
lus of  cholera  in  the  intestinal  excretion  of  apparently 
healthy  persons,  which  shows  that  they  were  protected  by 
the  resistance  of  their  tissues  to  the  bacilli.  Further  light 
has  been  thrown  on  this  question  by  the  researches  of  Mac- 
Fadyen,  who  has  pointed  out  that  suspensions  of  cholera 
bacilli  in  water  passed  through  the  stomach  untouched,  and 
were  thus  able  to  exert  their  evil  influence  in  other  parts  of 
the  alimentary  canal.  When,  however,  cholera  bacilli  were 
suspended  in  milk,  none  appeared  to  escape  the  germicidal 
action  of  the  gastric  juice.  The  explanation  of  this  is  prob- 
ably the  simple  one  that  the  stomach  reacted  with  its  secre- 
tion of  gastric  juice  only  to  food  (milk),  but  simply  passed 


84  BACl^ERIA 

the  water  on  into  the  lower  and  more  absorptive  parts  of  the 
alimentary  canal.  Such  a  condition  of  affairs  clearly  in- 
creases the  danger  of  water-borne  germs. 

THE   BACTERIOLOGY   OF   SEWAGE   AND    SEWAGE-POLLUTED 

WATERS 

It  will  not  be  needful  to  insist  upon  the  obvious  fact  that 
bacteria  abound  in  sewage.  Such  a  large  quantity  of  organic 
matter,  in  which  decomposition  is  constantly  taking  place, 
will  afford  an  almost  ideal  nidus  for  micro-organic  life. 
There  is  indeed  but  one  reason  why  such  a  medium  is  not 
absolutely  ideal  from  the  microbe's  point  of  view,  and  that 
reason  is,  that  in  sewage  the  vast  number  of  bacteria  present 
make  the  struggle  for  existence  exceptionally  keen.  Not 
only  are  the  numbers  incredibly  large,  but  we  also  find  a 
very  extensive  representation  of  species,  including  both 
saprophytes  and  parasites,  non-pathogenic  and  pathogenic. 
Not  infrequently  it  is  from  pollution  by  sewage  that  drink- 
ing water  is  contaminated  with  disease.  A  patient,  we  will 
say,  suffers  from  typhoid  fever.  The  specific  organism  has 
its  habitat  largely,  though  not  exclusively,  in  the  alimentary 
canal.  It  passes  out  in  the  excreta,  and  though  sometimes 
partially  disinfected,  may  escape  without  hindrance  into  the 
drains,  and  thus  to  the  sewer  or  cesspool.  How  often,  by 
means  of  direct  connection  or  by  percolation,  sewage,  from 
sewers  or  cesspools,  gains  access  to  drinking  water,  the 
history  of  typhoid  outbreaks  in  this  country  only  too  fully 
records. 

It  is  impossible  to  lay  down  any  exact  standard  of  the 
chemical  and  bacteriological  quality  of  sewage.  The  qual- 
ity will  differ  according  to  the  size  of  the  community,  the 
inclusion  or  otherwise  of  trade-waste  effluents,  the  addition 
of  rain-water,  and  other  like  physical  conditions.  Moreover, 
sewage  itself  when,  so  to  speak,  fully  formed  is  liable  to 
undergo  rapid  changes  owing  to  fermentation  and  the  com- 


BACTERIA   IN    WATER  85 

petition  of  micro-organisms.  It  is  clear  that  these  latter  are 
the  chief  agents  in  bringing  about  the  change,  because,  if 
sewage  be  placed  in  hermetically  sealed  flasks  and  sterilised 
by  heat,  it  is  found  that  no  change  occurs.  From  facts  such 
as  the  above  it  will  be  apparent  that  no  exact  standard  of 
chemical  or  bacterial  contents  is  possible.  Respecting  the 
chemical  condition  we  may  shortly  say  that  the  chief  char- 
acteristic of  sewage  is  its  enormous  amount  of  contained 
organic  matter  in  suspension  or  solution;  respecting  the 
bacterial  content  we  may  say  that  the  chief  species  of  the 
very  numerous  organisms  are  those  commonly  concerned  in 
fermentative  putrefaction.  London  crude  sewage  contains 
on  an  average  about  four  millions  of  micro-organisms  per 
cc.  Many  of  these  are  "  liquefying"  bacteria;  that  is  to 
say,  they  have  the  power  of  liquefying  gelatine,  which  is 
generally  one  of  the  features  of  putrefactive  species.  In 
considering  the  quality  of  the  bacteria  present  in  sewage,  a 
still  wider  field  of  research  opens  before  us.  For  though  we 
can  say  that,  roughly,  all  sewage  will  contain  probably  be- 
tween four  and  eight  millions  of  bacteria,  we  cannot  even 
lay  down  a  rough  standard  respecting  the  kinds  of  bacteria 
present  more  than  we  have  done  already  in  stating  that  a 
very  large  number  indeed  out  of  the  total  will  belong  to 
putrefactive  species. 

We  may,  however,  make  a  provisional  list  of  normal 
sewage  bacteria  1  as  follows : 

1  The  methods  adopted  for  making  a  quantitative  and  qualitative  examination 
of  sewage  are  precisely  analogous  to  those  used  in  milk  research.  Dilution 
with  sterilised  water  previous  to  plating  out  on  gelatine  in  Petri  dishes  is  essen- 
tial (i  cc.  to  10,000  cc.  of  sterile  water,  or  some  equally  considerable  dilution), 
otherwise  the  large  numbers  of  germs  would  rapidly  liquefy  and  destroy  the  film. 
Special  methods  must  be  used  for  the  isolation  of  special  organisms  ;  phenol- 
gelatine,  Eisner  medium,  indol  reaction,  "  shake"  cultures,  Parietti  broth,  etc., 
must  often  be  resorted  to  for  special  bacteria.  Spores  of  bacteria  may  always 
be  numerically  estimated  by  adding  the  suspected  water  or  sewage  to  gelatine, 
and  then  heating  to  80°  C.  for  ten  minutes  before  plating  out.  This  tempera- 
ture removes  the  bacilli,  but  leaves  the  spores  untouched. 


86 


BACTERIA 


1.  Bacillus  coli  communis  and  all  its  varieties  and  allies. 

2.  Proteus  vulgaris  and  the  various  protean  species. 

3.  B.  enteritidis  sporogenes  (Klein). 

4.  Liquefying  bacteria,  e.g.  ,  Bacillus  fluorescens  liquefa- 
dens,  B.  subtilis,  B.  mesentericus. 

5.  Non-liquefying  bacteria. 

6.  Sarcinae,  yeasts,  and  moulds. 


B.  ENTERITIDIS  SPOROGENES 


PROTEUS  VULGARIS 


We  have  not  included  in  the  above  inventory  any  patho- 
genic bacteria.  Doubtless  such  species  (e.g.,  typhoid ')  not 
infrequently  find  their  way  into  sewage.  But  they  are  not 
normal  habitants,  and  though  they  struggle  for  survival,  the 
keenness  of  competition  among  the  dense  crowds  of  sapro- 
phytes makes  existence  almost  impossible  for  them.  Nor 
can  they  expect  much  sympathy  from  us  in  the  difficulties 
of  life  which  fortunately  confront  them  in  sewage. 

Of  those  we  have  named  as  normally  present  it  is  unneces- 
sary to  speak  in  detail,  with  the  exception  of  the  newly 

1  The  bacilli  of  typhoid  can  live  in  crude  sewage  (Klein),  but  only  for  a  very 
short  period.  When  sewage  is  diluted  with  large  quantities  of  water  the  case 
is  very  different.  Bacillus  coli  flourishes  in  sewage. 


BACTERIA    IN    WATER  8/ 

discovered  anaerobe,  Bacillus  enteritidis  sporogenes  of  Klein.1 
This  bacillus  is  credited  to  be  a  causal  agent  in  diarrhoea,  and 
has  been  isolated  by  Dr.  Klein  from  the  intestinal  contents 
of  children  suffering  from  severe  diarrhoea,  and  from  adults 
having  cholera  nostras.  It  has  been  readily  detected  in 
sewage  from  various  localities,  and  also  in  sewage  effluents, 
after  sedimentation,  precipitation,  and  filtration.  Its  bio- 
logical characters  are  shortly  as  follows :  It  is  in  thickness 
somewhat  like  the  bacillus  of  symptomatic  anthrax,  thicker 
and  shorter  than  the  bacillus  of  malignant  oedema,  and 
standing  therefore  between  the  latter  and  anthrax  itself.  It 
is  motile  and  possesses  flagella,  but  has  no  threads.  It 
readily  forms  spores,  which  develop  as  a  rule  near  the  ends 
of  the  rods  and  are  thicker  than  the  bacilli.  It  is  stained  by 
Gram's  method.  In  various  media  (particularly  milk)  it 
produces  gas  rapidly.  It  is  an  anaerobe,  and  is  cultivated 
in  Buchner's  tubes.  A  recent  epidemic  of  diarrhoea  affect- 
ing 144  patients  in  St.  Bartholomew's  Hospital  was  traced 
to  milk  in  which  B.  enteritidis  was  present. 

Sewer  Air.  Though  not  of  material  importance  as  re- 
gards bacterial  treatment  of  sewage,  this  subject  calls  for 
some  remark.  For  long  it  has  been  known  that  air  polluted 
by  sewage  emanations  is  capable  of  giving  rise  to  various 
degrees  of  ill-health.  These  chiefly  affect  two  parts  of  the 
body;  one  is  the  throat,  and  the  other  is  the  alimentary 
canal.  Irritation  and  inflammation  may  be  set  up  in  both 
by  sewer  air.  Such  conditions  are  in  all  probability  pro- 
duced by  a  lowering  of  the  resistance  and  vitality  of  the 
tissues,  and  not  by  either  a  conveyance  of  bacteria  in  sewer 
air  or  any  stimulating  effect  upon  bacteria  exercised  by 
sewer  air.  What  evidence  we  have  is  against  such  factors. 
(See  p.  105.) 

Several  series  of  investigations  have  been  made  into  the 

1  Annual  Report  of  the  Medical  Officer  of  the  Local  Government  Board, 

1897-98,  p.  210. 


88  BACTERIA 

bacteriology  of  sewer  air,  amongst  others  by  Uffelmann, 
Haldane,  Laws,  and  Andrewes.  From  their  labours  we 
may  formulate  four  simple  conclusions : 

1.  The  air  of  sewers  contains  very  few  micro-organisms 
indeed,  sometimes  not  more  than  two  organisms  per  litre 
(Haldane),  and  generally  fewer  than  the  outside  air  (Laws 
and  Andrewes). 

2.  There  is  no  relationship  between  the  microbes  con- 
tained in  sewer  air  and  those  contained  in  sewage.      Indeed, 
there  is  a  marked  difference  which  forms  a  contrast  as  strik- 
ing as  it  is  at  first  sight  unexpected.     The  organisms  isol- 
ated from  sewer  air  are  those  commonly  present  in  the  open 
air.     Micrococci  and  moulds  predominate,  whereas  in  sew- 
age bacilli  are  most  numerous.     Liquefying  bacteria,  too, 
which  are  common  in  sewage,  are  extremely  rare  in  sewer 
air.     Bacillus  coli  communis,  which  occurs  in  sewage  from 
20,000  to  200,000  per  cc.,  is  altogether  absent  from  sewer  air. 

3.  Pathogenic  organisms  and  those  nearly  allied  to  them 
are  found  in  sewage,  but  absent  in  sewer  air.     Uffelmann 
isolated    the    Staphylococcus   pyogenes   aureus   (one    of   the 
organisms  of  suppuration),  but  such  a  species  is  exceptional 
in  sewer  air.     Hence,  though  sewer  air  is  popularly  held 
responsible  for  conveying  diphtheria  and  all  sorts  of  other 
virulent  bacteria,  there  is  up  to  the  present  no  evidence  of  a 
substantial    nature    in    support   of  such   views.     Sewer  air 
neither  conducts  pathogenic  organisms  nor  stimulates  the 
virulence  of  such. 

4.  Lastly,  only  when  there  is  splashing  in  the  sewage,  or 
when  bubbles  are  bursting  (Frankland),  is  it  possible  for 
sewage  to  part  with  its  contained  bacteria  to  the  air  of  the 
sewer. 

Whilst  we  cannot  here  enter  more  fully  into  an  account  of 
the  bacteria  found  in  sewage  or  of  their  functions,  it  is  ne- 
cessary to  remark  upon  one  distinguishing"  feature.  A  very 
large  number  of  sewage  bacteria  are  decomposing  and  denitri- 


BACTERIA   IN    WATER  89 

fying,  that  is  to  say,  breakers  down,  by  means  of  putrefac- 
tion, of  organic  compounds.  The  knowledge  of  this  fact 
has  recently  been  applied,  in  conjunction  with  oxidation,  to 
the  biological  treatment  of  sewage.  As  this  illustrates  in  a 
marked  degree  some  of  the  facts  we  have  dwelt  upon  in  con- 
sidering the  bacteriology  of  soil,  and  as  it  is  likely  that  the 
future  will  witness  a  still  wider  application  of  these  same 
facts,  it  will  be  necessary  to  refer  in  some  detail  to  the 
matter. 

Hitherto  there  has  been  adopted  one  of  four  methods  of 
treatment  of  sewage.  In  the  first  place,  in  towns  situated 
on  the  coast  the  sewage  has,  by  means  of  a  conduit,  been 
carried  out  to  sea.  It  is  clear  that  such  a  course,  which  is  in 
itself  open  to  criticism,  is  applicable  to  but  few  towns.  In 
the  second  place,  methods  of  chemical  treatment  have  been 
practised.  This  has  generally  been  of  the  nature  of  a 
"  precipitation  "  process.  Six  to  twelve  grains  of  quicklime 
have  been  added  to  each  gallon  of  sewage.  The  process  is 
simple  and  cheap,  but  it  does  not  remove  the  organic  matter 
in  solution.  On  the  one  hand,  it  does  not  produce  a  valu- 
able manure ;  on  the  other,  it  fails  to  purify  the  effluent.  A 
dozen  other  methods  have  been  tried,  but  all  based  on  the 
addition  of  chemical  substances  to  precipitate  or  change  the 
organic  matter  of  the  sewage.  Electrolysis,  too,  has  been 
proposed.  The  third  mode  adopted  in  the  past  has  been 
that  known  as  intermittent  downward  filtration.  This  may 
be  defined  as  "  the  concentration  of  sewage  at  short  inter- 
vals on  an  area  of  specially  chosen  porous  ground,  as  small 
as  will  absorb  and  cleanse  it,  not  excluding  vegetation,  but 
making  the  product  of  secondary  importance  ' '  (Metropolitan 
Sewage  Commission).  The  action  is  mechanical  and  bio- 
logical, that  is  to  say,  due  in  part  to  nitrification  by  bacteria 
in  the  upper  layers  of  soil.  The  fourth  plan  is  that  of  irri- 
gation, or  "  the  distribution  of  sewage  over  a  large  surface 
of  ordinary  agricultural  ground,  having  in  view  a  maximum 


go  BACTERIA 

growth  of  vegetation  (consistently  with  due  purification)  for 
the  amount  of  sewage  supplied."  Like  the  former,  there  is 
biological  influence  at  work  here,  though  in  a  less  degree. 
About  one  acre  is  required  for  every  hundred  persons  in  the 
population.  These  two  latter  modes  are  much  to  be  pre- 
ferred to  chemical  treatment,  yet  on  account  of  space  and 
management,  as  well  as  on  account  of  the  non-removal  of 
the  "  sludge,"  their  success  has  not  been  all  that  could 
be  desired.  Until  comparatively  recent  times  the  above 
methods  of  treating  sewage  were  the  only  ones  available. 

In  1 88 1  it  appears  that  M.  Louis  Mouras,  of  Vesoul  (Haute 
Saone),  published  an  account  of  a  hermetically  sealed, 
inodorous,  and  automatically  discharging  cesspool,  in  which 
sewage  was  anaerobically  broken  down  by  "  the  mysterious 
agents  of  fermentation."  This  is  the  first  record  we  have 
of  the  newly  applied  treatment  of  sewage  by  simply  allow- 
ing Nature  to  fulfil  her  function  by  means  of  bacteria.  We 
shall  most  easily  arrive  at  an  appreciation  of  the  recent  de- 
velopments of  the  process  in  England  by  describing  the 
so-  called  septic  tank  and  cultivation  beds. 

The  septic  tank  is  a  large  underground  vault  of  cemented 
brick,  having  a  capacity  of  thousands  of  gallons,  according 
to  the  population.  That  at  Exeter  has  a  capacity  of  53,800 
gallons,  and  takes  the  average  sewage  of  1 500  inhabitants  in 
twenty-four  hours.  Near  the  entrance  is  a  submerged  wall, 
seven  feet  from  the  entrance  and  twelve  inches  below  the 
surface  of  the  liquid  in  the  full  tank.  Within  this  are  caught, 
by  gravity,  gravel  and  such-like  deposits.  The  remaining 
solid  matter  of  the  sewage  becomes  deposited  in  the  tank  it- 
self. Both  in  the  sediment  at  the  bottom  of  the  tank  and  in 
the  thick  scum  on  the  surface  the  organic  compounds  are 
broken  down  and  made  soluble.  In  the  former  position  this  is 
accomplished  by  anaerobic  bacteria,  in  the  latter  on  the  sur- 
face by  aerobic  bacteria.  It  need  hardly  be  added  that  these 
are  denitrifying  and  putrefactive  bacteria,  and  that  those  at 


92  BACTERIA 

the  bottom  of  the  tank  perform  greater  service  than  those 
at  the  top.  When  the  liquid  sewage  passes  out  of  the  tank 
it  differs  from  the  crude  sewage  which  enters  the  tank  in  the 
following  particulars :  (a)  The  gravel  and  particulate  debris 
have  been  removed ;  (b)  the  organic  solids  in  suspension  are 
so  greatly  diminished  that  they  are  almost  absent ;  (c)  there 
is  an  increase  of  organic  matter  in  solution  ;  (  d)  the  sewage 
is  darker  in  colour  and  more  opalescent ;  (e)  compounds  like 
albuminoid  ammonia,  urea,  etc.,  have  been  more  or  less 
completely  broken  down,  and  reappear  in  elementary  con- 
ditions, like  ammonia,  methane,  carbon  dioxide,  and  sul- 
phuretted hydrogen.  These  latter  bodies  may  be  in  solution 
or  may  have  escaped  as  gas. 

The  cultivation  beds  are  four  or  five  filters,  to  which  the 
sewage  from  the  tank  flows  in  such  a  manner  as  to  produce 
a  weir.  By  an  automatic  arrangement  the  fluid  is  distributed 
to  each  filter  in  turn.  When  the  second  filter  is  full  the 
first  is  discharged,  and  remains  empty  during  the  time  that 
the  third  and  fourth  are  being  filled.  Each  filter  is  thus 
full,  say,  about  six  hours,  and  has  from  ten  to  twelve  hours' 
rest.  These  filter-beds  (at  Exeter)  have  an  area  of  eighty 
square  yards  and  a  depth  of  five  feet ;  collecting  drains  are 
laid  on  the  bottom  of  the  filters,  joining  main  collectors, 
the  latter  terminating  in  discharging  wells.  The  filtrant  is 
broken  furnace  clinker  or  broken  coke. 

The  changes  occurring  in  these  filters  are  of  the  nature  of 
oxidation,  with  the  result  that  the  proportion  of  the  oxidised 
nitrogen  increases  (as  nitrites  and  nitrates),  the  ammonia 
becomes  less,  and  the  total  solids  and  organic  nitrogen 
almost  disappear.  It  will  thus  be  seen  that  the  work  of 
these  filters  is  not  merely  a  straining  action.  It  is  true  that 
particulate  matter  in  the  effluent  from  the  tank  is  caught  on 
the  surface  by  the  film  (resulting  from  previous  effluents), 
but  the  real  work  of  the  bed  is  nitrification,  an  oxidation  of 
ammonia  into  nitrites  and  nitrates.  This  change  obviously 


BACTERIA   IN    WATER  93 

begins  when  the  tank  effluent  flows  over  the  "  weir  "  on  to 
the  filter-beds,  and  the  oxygen  thus  obtained  by  the  effluent 
is  carried  down  in  solution  into  the  coke-breeze.  Upon  the 
surface  of  the  filtrant  are  oxidising  bacteria.  When  the 
effluent  is  on  the  bed  they  oxidise  its  contained  products; 
when  the  bed  is  empty  and  "  resting  "  they  oxidise  carbon. 
An  advantage  arising  from  the  periodical  emptying  and 
filling  of  the  filter  is  that  the  products  of  decomposition 
which  would  eventually  inhibit  the  action  of  the  aerobic 
bacteria  are  washed  away,  and  pass  into  the  nearest  stream, 
where  they  become  absolutely  innocuous. 

The  "  filter  "  is  more  correctly  termed  a  cultivation  bed, 
for  its  purpose  is  to  furnish  a  very  large  surface  upon  which 
the  nitrifying  organisms  present,  as  we  have  seen,  in  all  soils, 
may  flourish,  and  thus  feeding  upon  the  organic  matter  of 
the  sewage,  may  perform  their  function  of  oxidation. 

It  is  not  possible  to  lay  down  exact  limits  as  to  where 
denitrification  ends  and  oxidation  begins.  To  a  certain 
extent,  and  in  varying  degree,  they  overlap  each  other. 
But  roughly  we  may  say  that  in  the  tank  there  is  a  breaking 
down  (denitrification  and  decomposition)  and  in  the  filter- 
beds  a  building  up  (nitrification).  The  case  is  precisely 
parallel  to  similar  changes  occurring  in  soil,  and  which  we 
have  dealt  with  elsewhere.  The  advantage  indeed  of  this 
biological  treatment  of  sewage  is  that  it  exactly  follows  the 
processes  of  ^nature,  in  contradistinction  to  the  mechanical 
and  chemical  methods  hitherto  adopted. 

At  Sutton  and  some  other  places  the  same  principles  are 
applied, — that  is  to  say,  bacterial  filtration, — but  there  is  no 
tank.  A  metal  screen  in  some  measure  takes  its  place,  and 
holds  back  solid  matter  from  being  carried  on  to  the  beds. 
The  filtrant  is  burnt  clay,  and  it  is  forked  over  occasionally 
to  let  in  oxygen.  The  crude  sewage  is  run  over  the  top 
of  the  burnt  ballast,  where  it  is  left  for  two  or  three  hours. 
It  is  then  slowly  run  off  on  to  a  finer  filter,  where  it  also 


94 


BA  CTERIA 


stays  two  hours.     Thence  the  effluent  is  run  into  the  stream. 
It  must  be  admitted  that  the  bacterial  treatment  of  sew- 
age, though  exhibiting  such  excellent  results  where  it  has 
been  given  a  fair  trial,  is  still  in  a  probationary  stage.     It 


Metal; 
Screen! 


, Conduit  to  Beds 


-Filter  Bed  full  of 
Burnt  Clay 

Exit  Pipe 


Filter  Bed  empty 
of  Filtrant,  showing 

Exit  Pipe  at  the 

bottom 


FILTER-BEDS 

As  Used  at  Sutton 

appears  to  stand  on  reason.  The  sludge  of  previous  methods 
is  avoided.  The  sewage  is  entirely  broken  down,  and  the  ef- 
fluent is  a  comparatively  pure  one,  yet  taking  back  nitrogen, 
as  nitrate,  to  the  soil.  The  whole  change,  indeed,  in  the 
opinion  of  Dr.  Dupre,  is  more  effective  and  radical  than  in 
chemical  treatment.  Further,  it  has  been  tested  as  regards 


BACTERIA   IN    WATER  95 

its  action  upon  the  pathogenic  bacilli — those  of  tubercle  and 
typhoid — with  the  result  that  these  infective  bacteria  have 
been  completely  destroyed.  It  appears  that  such  destruc- 
tion of  infective  germs  occurs  in  the  tank,  and  depends  in 
degree  upon  the  rapidity  with  which  sewage  is  passed 
through  the  tank.  The  cultivation  beds  also  have  an 
inimical  effect  upon  infective  bacteria.  Hence  the  final 
effluent  is  practically  germ-free  as  regards  pathogenic 
organisms. 


CHAPTER  III 

BACTERIA  IN  THE  AIR 
METHODS   OF   EXAMINING  AIR   FOR   BACTERIA 

THE  basis  of  the  usual  methods  in  practice  is  to  pass  air 
over  or  through  some  nutrient  medium.  By  this 
means  the  contained  organisms  are  waylaid,  and  rinding 
themselves  under  favourable  conditions  of  pabulum,  tem- 
perature, and  moisture,  commence  active  growth,  and  thus 
reveal  themselves  in  characteristic  colonies.  These  are 
examined,  as  directed  on  page  43,  by  the  microscope  and 
sub-culture.  Quantitative  estimation  is  not  generally 
made,  as  a  fixed  standard  is  even  less  a  possibility  than  in 
milk  and  soil.  Returns  of  the  number  of  bacteria  in  the 
sample  taken  may  be  made  for  the  sake  of  information,  but 
little  or  no  conclusion  of  value  can  be  drawn  from  such 
data.  The  standard  recognised  in  Europe  is  the  cubic  metre, 
and  one  may  speak,  for  example,  of  the  air  of  a  room  con- 
taining 500,  1000,  or  3000  germs  per  cubic  metre. 

The  following  are  the  chief  methods : 

i.  Pouchet's  Aeroscope.  This  apparatus  was  in  use  some 
time  ago  in  France,  and  by  its  means  all  the  solid  matter  of 
a  given  quantity  of  air  was  drawn  through  an  air-tight  glass 
tube  by  aspiration  and  made  to  impinge  upon  a  small  plate 
of  glycerine.  The  air  escaped  to  the  aspirator  at  the  sides, 
leaving  upon  the  glycerine  plate  only  its  particulate  matter. 
This  remnant  could  then  be  examined. 

96 


BACTERIA   IN   THE  AIR 


97 


2.  Koch  adopted  the  simplest  of  all  the  culture  methods, 
viz.,  exposing  a  plate  of  gelatine  or  agar  for  a  longer  or 
shorter  time  to  the  air  of  which  examination  is  desired.     By 
gravity  the  suspended  bacteria  fall  on  the  plate  and  start 
growth.    As  a  matter  of  quantitative  exactitude,  this  method 
is  not   to   be   recommended,    but   it   frequently  proves  an 
excellent  method  for  qualitative  estimation. 

3.  The  Method  of  MiqueL     Pasteur  was  the  first  to  analyse 
air  by  the  culture  method,  and  he  adopted  a  plan  which  in 
principle  is  washing  the  air  in  some  fluid  culture  medium 
which  will  retain  all  the  particulars  matter,  which  may  then 
be   cultured   directly   or  sub-cultured    into  any  favourable 
medium. 

Miquel  has  contrived  a  simple  piece  of  apparatus  for  the 
carrying  out  of  this  principle.  It  consists  of  a  flask  with  a 
central  tube  through  its  own  neck 
for  the  entrance  of  the  air.  On 
one  side  of  the  flask  is  a  tube  to 
be  connected  with  the  aspirator, 
on  the  other  side  of  the  flask  a 
tube  through  which  to  pour  off 
the  contained  fluid  at  the  end  of 
the  process.  In  the  flask  are 
placed  30  cc.  of  sterilised  water 
(or,  indeed,  if  it  be  preferred, 
sterilised  broth).  The  entrance 
tube  is  now  unplugged,  and  the 
aspirator  draws  through  a  fair 
sample  of  the  air  in  the  room  (say 
ten  litres).  This  air  perforce 
passes  through  the  water  and  by 
the  exit  tube  to  the  aspirator,  and 
is  thereby  washed,  leaving  behind 
in  the  water  all  its  bacteria.  The 
aspiration  is  then  stopped,  and  the 


MIQUEL'S  FLASK 


98  BACTERIA 

entrance  tube  closed.  The  water  (plus  bacteria)  is  now 
poured  out  into  test-tubes  of  media  or  plated  out  on  Petri's 
dishes.  Provided  the  apparatus  has  been  absolutely  steril- 
ised, and  that  the  water  was  also  sterile,  any  colonies 
developing  upon  the  Petri  dish  are  composed  of  micro- 
organisms from  the  air  examined. 

4.  The  Method  of  Hesse.  This  method  is  somewhat  akin 
to  Pouchet's  aeroscope,  but  is  in  addition  a  culture  method. 
Hesse's  tube  is  about  2  feet  long  and  i£  inches  bore  through- 
out. At  one  end  is  an  india-rubber  stopper  bored  for  a  glass 
tube  to  the  aspirator.  The  other  end  is  open.  Before  using, 
the  tube  is  sterilised,  and  40  or  50  cc.  of  sterilised  gelatine 
replaced  in  it.  The  tube  is  now  rapidly  rotated  in  a  groove 
on  a  block  of  ice  or  under  a  cold-water  tap,  and  by  this 
simple  means  the  gelatine  becomes  fixed  and  forms  a  layer 
inside  the  tube  throughout.  We  have  therefore,  so  to 
speak,  a  tube  of  glass  with  a  tube  of  gelatine  inside  it.  The 
apparatus  is  now  ready  for  use.  It  is  fixed  on  the  tripod, 
and  fifteen  litres  of  air  are  drawn  through,  and  the  tube  is 
properly  plugged  and  incubated  at  room  temperature.  In 
a  day  or  two  days  the  colonies  appear  upon  the  gelatine. 
They  are  most  numerous  generally  in  the  first  part  of  the 
tube,  and  might  be  roughly  estimated  as  follows : 

15  litres  of  air,  6  colonies. 

.'.-^g-  X  10,000  =  4000  aerobic  bacteria  in  the  cubic  metre. 

The  disadvantages  of  this  process  are  that  dried  gelatine 
does  not  catch  germs  like  the  broth  cultures  of  Pasteur  or 
Miquel,  and  that  many  organisms  are  able  to  go  straight 
through  the  tube,  and  failing  to  be  deposited,  pass  out  at 
the  aspirator  exit,  and  thus  are  neither  caught  nor  counted. 
The  Hesse  tube  is  generally  used  in  practice  with  a  pump 
consisting  of  two  flasks  and  a  double-way  india-rubber  tube. 
The  flasks  have  a  capacity  for  one  litre  of  water.  By  a 
simple  adaptation  it  is  possible  to  secure  siphon  action,  and 


BACTERIA    IN   THE  AIR  99 

hence  measure  with  considerable  exactitude  the  amount  of 
air  passing  through  the  tube. 

5.  Methods  of  Filtration.  To-day  most  of  the  above 
methods  have  been  discarded,  with  the  exception,  perhaps, 
of  Miquel's  and  modifications  thereof. 

Frankland,  Petri,  Pasteur,  Sedgwick,  and  others  have 
suggested  the  adoption  of  methods  of  filtration.  These 
depend  upon  catching  the  organisms  contained  in  the  air  by 
filtering  them  through  sterilised  sand  or  sugar,  and  then 
examining  these  media  in  the  ordinary  way.  Many  differ- 
ent kinds  of  apparatus  have  been  invented.  Petri  aspirates 
through  a  glass  tube  containing  sterilised  sand,  which  after 
use  is  distributed  in  Petri  dishes  and  covered  with  gelatine. 
The  principal  objection  to  this  method  is  the  presence  of  the 
opaque  particles  of  sand  in  and  under  the  gelatine.  Prob- 
ably it  was  this  which  suggested  the  use  of  soluble  filters 
like  sugar.  Pasteur  introduced  the  principle,  and  Frankland 
and  others  have  followed  it  out.  The  apparatus  most  largely 
used  is  that  known  as  Sedgwick's  Tube.  This  consists  of 
a  comparatively  small  glass  tube,  about  a  foot  long.  Half 


J 


SEDGWICK'S  SUGAR-TUBE 

of  it  has  a  bore  of  2.5  cm.,  and  the  other  half  a  bore  of  .5 
cm.  It  is  sterilised  at  150°  C,  after  which  the  dry,  finely 
granulated  cane-sugar  is  inserted  in  such  a  way  as  to  occupy 
an  inch  or  more  of  the  narrow  part  of  the  tube  next  the 
wide  part.  Next  to  it  is  placed  a  wool  plug,  and  the  whole 
is  again  sterilised  at  130°  C.  for  two  hours,  care  being  taken 
that  the  sugar  does  not  melt.  After  sterilisation  an  india- 
rubber  tube  is  fixed  to  the  end  of  the  narrow  portion,  and 
thus  it  is  attached  to  the  aspirator.  The  measured  quantity 
(5-20  litres)  of  air  is  drawn  through,  and  any  particulate 


100 


BACTERIA 


matter  is  caught  in  the  sugar.  Warm,  nutrient  gelatine 
(10-15  cc.)  is  now  poured  into  the  broad  end  of  the  tube, 
and  by  means  of  a  sterilised  stilette  the  sugar  is  pushed 
down  into  the  gelatine,  where  it  quickly  dissolves.  We 
have  now  in  the  gelatine  all  the  micro-organisms  in  the  air 


SEDGWICK'S  TUBE 

Fixed  upon  Tripod  for  Air  Examination 


which  has  been  drawn  through  the  tube.  After  plugging 
with  wool  at  both  ends,  the  tube  is  rolled  on  ice  or  under 
a  cold-water  tap  in  order  to  fix  the  gelatine  all  round  the  in- 
ner wall  of  the  tube,  which  is  incubated  at  room  tempera- 


BACTERIA    IN    THE   AIR  IOI 

ture.     In  a  day  or  two  the  colonies  appear,  and   may   be 
examined. 

Micro-organisms  in  the  Air.  Schwann  was  one  of  the  first 
to  point  out  that  when  a  decoction  of  meat  is  effectually 
screened  from  the  air,  or  supplied  solely  with  calcined  air, 
putrefaction  does  not  set  in.  Helmholtz  and  Pasteur  con- 
firmed this,  but  it  may  be  said  with  some  truth  that  Schwann 
originated  the  germ  theory,  and  Lister  applied  it  in  the 
treatment  of  wounds.  Lister  believed  that  if  he  could  sur- 
round wounds  with  filtered  air  the  results  would  be  as  good 
as  if  they  were  shut  off  from  the  air  altogether. 

It  was  Tyndall 1  who  first  laid  down  the  general  principles 
upon  which  our  knowledge  of  organisms  in  the  air  is  based. 
That  the  dust  in  the  air  was  mainly  organic  matter,  living 
or  dead,  was  a  comparatively  new  truth ;  that  epidemic  dis- 
ease was  not  due  to  "  bad  air  "  and  "  foul  drains,"  but  to 
germs  conveyed  in  the  air,  was  a  prophecy  as  daring  as  it 
was  correct.  From  these  and  other  like  investigations  it 
came  to  be  recognised  that  putrefaction  begins  as  soon  as 
bacteria  gain  an  entrance  to  the  putrefiable  substance,  that 
it  progresses  in  direct  proportion  to  the  multiplication  of 
bacteria,  and  that  it  is  retarded  when  they  diminish  or  lose 
vitality. 

Tyndall  made  it  clear  that  both  as  regards  quantity  and 
quality  of  micro-organisms  in  the  air  there  neither  is  nor 
can  be  any  uniformity.  They  may  be  conducted  on  par- 
ticles of  dust — "  the  raft  theory  " — but  being  themselves 
endowed  with  a  power  of  flotation  commensurate  with  their 
extreme  smallness  and  the  specific  lightness  of  their  com- 
position, dust  as  a  vehicle  is  not  really  requisite.  Never- 
theless the  estimation  of  the  amount  of  dust  present  in  a 
sample  of  air  is  a  very  good  index  of  danger.  It  is  to  Dr. 
Aitken  that  we  are  indebted  for  devising  a  method  by  which 
we  can  measure  dust  particles  in  the  air,  even  though  they 

1  John  Tyndall,  F.R.S.,  Floating  Matter  of  the  Air. 


102  BACTERIA 

be  invisible.  His  ingenious  experiments,  reported  in  the 
Transactions  of  the  Royal  Society  of  Edinburgh  (vol.  xxxv.), 
have  demonstrated  that  by  supersaturation  of  air  the  invis- 
ible dust  particles  may  become  visible.  As  is  now  well 
known,  Dr.  Aitken  has  been  able  to  prove  that  fogs,  mists, 
and  the  like  do  not  occur  in  dust-free  air,  and  are  due  to 
condensation  of  moisture  upon  dust  particles.  But  it  should 
be  remembered  that,  though  dust  forms  a  vehicle  for  bac- 
teria, dusty  air  is  often  comparatively  free  from  bacteria. 
Hence,  after  all,  the  necessary  conditions  for  dissemination 
of  bacteria  in  air  are  two,  namely,  some  degree  of  air-cur- 
rent and  dry  surfaces. 

This  latter  condition  is  one  of  essential  importance.  Bac- 
teria cannot  leave  a  moist  surface  either  under  evaporation 
or  by  means  of  air-currents.1  Only  when  there  is  consider- 
able molecular  disturbance,  such  as  splashing,  can  there 
possibly  be  microbes  transmitted  to  the  surrounding  air. 
This  fact,  coupled  with  the  influence  of  gravitation,  is  the 
reason  why  sewer  gas  and  all  air  contained  within  moist 
perimeters  is  almost  germ-free;  whereas  from  dry  surfaces 
the  least  air-current  is  able  to  raise  countless  numbers  of 
organisms.  Quite  recently  this  principle  has  been  admir- 
ably illustrated  in  two  series  of  investigations  made  upon 
expired  and  inspired  air.  In  a  report  to  the  Smithsonian 
Institution  of  Washington  (1895)  upon  the  composition  of 
expired  air,  it  is  concluded  that  "  in  ordinary  quiet  respira- 
tion no  bacteria,  epithelial  scabs,  or  particles  of  dead  tissue 
are  contained  in  the  expired  air.  In  the  act  of  coughing  or 
sneezing  such  organisms  or  particles  may  probably  be  thrown 
out."  The  interior  of  the  cavity  of  the  mouth  and  external 
respiratory  tract  is  a  moist  perimeter,  from  the  walls  of 
which  no  organisms  can  rise  except  under  molecular  dis- 

1  Flttgge  has  lately  attempted  to  demonstrate  that  an  air  current  having  a 
velocity  of  four  metres  per  second  can  remove  bacteria  from  surfaces  of  liquids 
by  detaching  drops  of  the  liquid  itself. 


BACTERIA   IN    THE   AIR  103 

turbance.  The  position  is  precisely  analogous  to  the 
germ-free  sewer  air  as  established  by  Messrs.  Laws  and  An- 
drewes  for  the  London  County  Council.  The  popular  idea 
that  infection  can  be  "  given  off  by  the  breath  "  is  contrary 
to  the  laws  of  organismal  pollution  of  air.  The  required 
conditions  are  not  fulfilled,  and  such  breath  infection  must 
be  of  extremely  rare  occurrence.  The  air  can  only  be  infec- 
tive when  filled  with  organisms  arising  from  dried  surfaces. 

The  other  series  of  investigations  were  conducted  by  Drs. 
Hewlett  and  St.  Clair  Thompson,  and  dealt  with  the  fate 
of  micro-organisms  in  inspired  air  and  micro-organisms 
in  the  healthy  nose.  They  estimated  that  from  1500 
to  14,000  bacteria  were  inspired  every  hour.  Yet,  as  we 
have  pointed  out,  expired  air  contains  practically  none  at 
all.  It  is  clear,  then,  that  the  inspired  bacteria  are  de- 
tained somewhere.  Lister  has  pointed  out,  from  observ- 
ation on  a  pneumo-thorax  caused  by  a  wound  of  the  lung 
by  a  fractured  rib,  that  bacteria  are  arrested  before  they 
reach  the  air-cells  of  the  lung;  hence  it  is  at  some  inter- 
mediate stage  that  they  are  detained.  Hewlett  and  Thom- 
son examined  the  mucus  from  the  wall  of  the  trachea,  and 
found  it  germ-free.  It  was  only  when  they  reached  the 
mucous  membrane  and  moist  vestibules  and  vibrissae  of  the 
nose  that  they  found  bacteria.  Here  they  were  present  in 
abundance.  The  ciliated  epithelium,  the  moist  mucus,  and 
the  bactericidal  influence  of  the  wandering  or  "  phagocyte  " 
cells  probably  all  contribute  to  their  final  removal.1 

There  can  be  no  doubt  that  the  large  number  of  bacteria 
present  in  the  moist  surfaces  of  the  mouth  is  the  cause  of  a 
variety  of  ailments,  and  under  certain  conditions  of  ill- 
health  organisms  may  through  this  channel  infect  the  whole 

1  Hewlett  and  Thomson  graphically  demonstrated  the  bactericidal  power  of 
the  nasal  mucous  membrane  by  noting  the  early  removal  of  Bacillus prodigiosus, 
which  had  been  purposely  placed  on  the  healthy  Schneiderian  membrane  of  the 
nose. 


104  BACTERIA 

body.  Dental  caries  will  occur  to  everyone's  mind  as  a  dis- 
ease possibly  due  to  bacteria.  As  a  matter  of  fact,  probably 
acids  (due  to  acid  secretion  and  acid  fermentation)  and 
micro-organisms  are  two  of  the  chief  causes  of  decay  of 
teeth.  Defects  in  the  enamel,  inherent  or  due  to  injury, 
retention  of  debris  on  and  around  the  teeth,  and  certain 
pathological  conditions  of  the  secretion  of  the  mouth  are 
predisposing  causes,  which  afford  a  suitable  nidus  for  putre- 
factive bacteria.  The  large  quantity  of  bacteria  which  a 
decayed  tooth  contains  is  easily  demonstrated. 

From  the  two  series  of  experiments  which  we  have  now 
considered  we  may  gather  the  following  facts : 

(a)  That  air  may  contain  great  numbers  of  bacteria  which 
may  be  readily  inspired. 

(b)  That  in  health  those  inspired  do  not  pass  beyond  the 
moist  surface  of  the  nasal  and  buccal  cavities. 

(c)  That  here  there  are  various  influences  of  a  bactericidal 
nature  at  work  in  defence  of  the  individual. 

(d)  That  expired  air  contains,  as  a  rule,  no  bacteria  what- 
ever. 

The  practical  application  of  these  things  is  a  simple  one. 
To  keep  air  free  from  bacteria,  the  surroundings  must  be 
moist.  Strong  acids  and  disinfectants  are  not  required. 
Moisture  alone  will  be  effectual.  Two  or  three  examples  at 
once  occur  to  the  mind. 

Anthrax  spores  are  conveyed  from  time  to  time  from 
dried  infected  hides  and  skins  to  the  hands  or  bodies  of 
workers  in  warehouses  in  Bradford  and  other  places.  If  the 
surroundings  were  moist,  and  the  hides  moist,  anthrax 
spores  and  all  other  bacteria  would  not  remain  free  in  the 
air. 

The  bacilli  or  spores  of  tubercle  present  in  sputum  in  great 
abundance  cannot,  by  any  chance  whatever,  infect  the  air 
until,  and  unless,  the  sputum  dries.  So  long  as  the  ex- 
pectorated matter  remains  on  the  pavement  or  handkerchief 


BACTERIA   IN   THE  AIR  1 05 

wet,  the  surrounding  air  will  contain  no  bacilli  of  tubercle. 
But  when  in  the  course  of  time  the  sputum  dries,  then  the 
least  current  of  air  will  at  once  infect  itself  with  the  dried 
spores  and  bacilli. 

Typhoid  Fever,  too,  occupies  the  same  position.  Only 
when  the  excrement  dries  can  the  contained  bacteria  infect 
the  air.  It  is  of  course  well  known  that  the  common  chan- 
nel of  infection  in  typhoid  fever  is  not  the  air,  whereas 
the  reverse  holds  true  of  tuberculosis.  The  writer  recently 
obtained  some  virulent  typhoid  excrement,  and  placed  it 
in  a  shallow  glass  vessel  under  a  bell-jar,  with  similar  vessels 
of  sterilised  milk  and  of  water,  all  at  blood-heat.  So  long 
as  the  excrement  remained  moist,  even  though  it  soon  lost 
its  more  or  less  fluid  consistence,  the  milk  and  water  re- 
mained uninfected.  But  when  the  excrement  was  com- 
pletely dried  it  required  but  a  few  hours  to  reveal  typhoid 
bacilli  in  the  more  absorptive  fluid,  milk,  and  at  a  later 
stage  the  water  also  showed  clear  signs  of  pollution.  This 
evidence  points  in  the  same  direction  as  that  which  has 
gone  before.  If  the  excrement  of  patients  suffering  from 
typhoid  dries,  the  air  will  become  infected;  if,  on  the  other 
hand,  it  passes  in  a  moist  state  into  the  sewer,  even  though 
untreated  with  disinfectants,  all  will  be  well  as  regards  the 
surrounding  air.- 

Before  passing  on  to  consider  other  matters  concerning 
organisms  in  the  air,  we  may  draw  attention  to  some  in- 
teresting observations  recorded  by  Mr.  S.  G.  Shattock l  on 
the  negative  action  of  sewer  air  in  raising  the  toxicity  of 
lowly  virulent  bacilli  of  diphtheria.  Some  direct  relation- 
ship, it  has  been  surmised,  exists  between  breathing  sewer 
air  and  "  catching  "  diphtheria.  Clearly  it  cannot  be  that 
the  sewer  air  contains  the  bacillus.  But  some  have  sup- 
posed that  the  sewer  air  has  had  a  detrimental  effect  by 
increasing  the  virulent  properties  of  bacilli  already  in  the 

1  Pathological  Society  of  London,   Transactions,  1897. 


106  BACTERIA 

human  tissues.  Two  cultivations  of  lowly  virulent  bacilli 
were  therefore  grown  by  Mr.  Shattock  in  flasks  upon  a 
favourable  medium  over  which  was  drawn  sewer  air.  This 
was  continued  for  two  weeks  or  five  weeks  respectively.  Yet 
no  increased  virulence  was  secured.  Such  experiments  re- 
quire ample  confirmation,  but  even  from  this  it  will  be  seen 
that  sewer  air  does  not  necessarily  have  a  favouring  influence 
upon  the  virulence  of  the  bacilli  of  diphtheria. 

It  should  be  noted  that  the  bacilli  of  diphtheria  are  cap- 
able of  lengthened  survival  outside  the  body,  and  are  readily 
disseminated  by  very  feeble  air-currents.  The  condition 
necessary  for  their  existence  outside  the  body  for  any  period 
above  two  or  three  days  is  moisture.  Dried  diphtheria 
bacilli  soon  lose  their  vitality.  It  is  probably  owing  to  this 
fact  that  the  disease  is  not  as  commonly  conveyed  by  air  as, 
for  example,  tubercle.1 

The  influence  of  gravity  upon  bacteria  in  the  air  may  be 
observed  in  various  ways,  in  addition  to  its  action  within  a 
limited  area  like  a  sewer  or  a  room.  Miquel  found  in  some 
investigations  in  Paris  that,  whereas  on  the  Rue  de  Rivoli 
750  germs  were  present  in  a  cubic  metre,  yet  at  the  summit 
of  the  Pantheon  only  28  were  found  in  the  same  quantity 
of  air.  At  the  tops  of  mountains  air  is  germ-free,  and  bac- 
teria increase  in  proportion  to  descent.  As  Tyndall  has 
pointed  out,  even  ultra-microscopic  cells  obey  the  law  of 
gravitation.  This  is  equally  true  in  the  limited  areas  of  a 
laboratory  or  warehouse  and  in  the  open  air. 

The  conditions  which  affect  the  number  of  bacteria  in  the 
air  are  various.  After  a  fall  of  rain  or  snow  they  are  very 
markedly  diminished  ;  during  a  dry  wind  they  are  increased. 
In  open  fields,  free  from  habitations,  they  are  fewer,  as 
would  be  expected,  than  in  the  vicinity  of  manufactories, 
houses,  or  towns.  A  dry,  sandy  soil  or  a  dry  surface  of 
any  kind  will  obviously  favour  the  presence  of  organisms  in 

1  Annali  d"1  Igiene  Sperimetitale,  vol.  v.  (1895),  fasc.  4. 


BACTERIA   IN   THE  AIR  IO/ 

the  air.  Frankland  found  that  fewer  germs  were  present 
in  the  air  in  winter  than  in  summer,  and  that  when  the 
earth  was  covered  with  snow  the  number  was  greatly  re- 
duced. Miquel  and  Freudenreich  have  declared  that  the 
number  of  atmospheric  bacteria  is  greater  in  the  morning 
and  evening  between  the  hours  of  six  and  eight  than  during 
the  rest  of  the  day.  But  we  venture  to  express  the  hope 
that  such  coincidental  facts  may  not  be  exalted  into  prin- 
ciples. 

There  is  no  numerical  standard  for  bacteria  in  the  air  as 
there  is  in  water.  The  open  air  possibly  averages  about  250 
per  cubic  metre.  On  the  seacoast  this  number  would  fall 
to  less  than  half;  in  houses  and  towns  it  would  rise  accord- 
ing to  circumstances,  and  frequently  in  dry  weather  reach 
thousands  per  cubic  metre.  When  it  is  remembered  that 
air  possesses  no  pabulum  for  bacteria  as  do  water  and  milk,  it 
will  be  understood  that  bacteria  do  not  live  in  the  air.  They 
are  only  driven  by  air-currents  from  one  dry  surface  to 
another.  Hence  the  quality  and  quantity  of  air  organisms 
depend  entirely  upon  environment  and  physical  conditions. 
In  some  researches  which  the  writer  made  into  the  air  of 
workshops  in  Soho  in  1896,  it  was  instructive  to  observe 
that  fewer  bacteria  were  isolated  by  Sedgwick's  sugar-tube 
in  premises  which  appeared  to  the  naked  eye  polluted  in  a 
large  degree  than  in  other  premises  apparently  less  con- 
taminated. In  the  workroom  of  a  certain  skin-curer  the 
air  was  densely  impregnated  with  particles  from  the  skin, 
yet  scarcely  a  single  bacterium  was  isolated.  In  the  polish- 
ing-room  of  a  well-known  hat  firm,  in  which  the  air  appeared 
'  to  the  naked  eye  to  be  pure,  and  in  which  there  was  ample 
ventilation,  there  were  found  four  or  five  species  of  sapro- 
phytic  bacteria.  Quite  recently  Mr.  S.  R.  Trotman,  public 
analyst  for  the  city  of  Nottingham,  estimated  the  bacterial 
quality  of  the  air  of  the  streets  of  that  town  during  "  the 
goose  fair  "  held  in  the  autumn.  He  used  a  modification 
of  Hesse's  apparatus  in  which  the  gelatine  is  replaced  by 


108  BACTERIA 

glycerine.  The  air  was  slowly  drawn  through  and  measured 
in  the  usual  way.  Sterilised  water  was  then  added  to  bring 
the  glycerine  to  a  known  volume,  the  liquid  thoroughly 
mixed,  and  a  series  of  gelatine  and  agar  plates  made  with 
quantities  varying  from  o.  I  to  2  cc.  By  this  method  a  large 
number  of  bacteria  were  detected  in  this  particular  invest- 
igation, including  Staphylococcus  pyogenes  aureus  et  albus, 
the  common  Bacillus  subtilis,  and  B.  coli  communis.1 

During  a  six  years'  investigation  the  air  of  the  Montsouris 
Park  yielded,  according  to  Miquel,  an  average  of  455  bac- 
teria per  cubic  metre.  In  the  middle  of  Paris  the  average 
per  cubic  metre  was  nearly  4000.  Fliigge  accepts  100  bac- 
teria per  cubic  metre  as  a  fair  average.  From  this  fact  he 
estimates  that  "  a  man  during  a  lifetime  of  seventy  years  in- 
spires about  25,000,000  bacteria,  the  same  number  contained 
in  a  quarter  of  a  litre  of  fresh  milk."  *  Many  authorities 
would  place  the  average  much  below  100  per  cubic  metre, 
but  even  if  we  accept  that  figure  it  is  at  once  clear  how 
relatively  small  it  is.  This  is  due,  as  we  have  mentioned, 
to  sunlight,  rain,  desiccation,  dilution  of  air,  moist  surfaces, 
etc.  So  essentially  does  the  bacterial  content  of  air  depend 
upon  the  facility  with  which  certain  bacteria  withstand  dry- 
ing that  Dr.  Eduardo  Germano  3  has  addressed  himself  first 
to  drying  various  pathogenic  species  and  then  to  mixing  the 
dried  residue  with  sterilised  dust  and  observing  to  what 
degree  the  air  becomes  infected.  Typhoid  appears  to  with- 
stand comparatively  little  dessication,  without  losing  its 
virulence.  Nevertheless,  it  is  able  to  retain  vitality  in  a 
semi-dried  condition,  and  it  is  owing  to  this  circumstance 
in  all  probability  that  it  possesses  such  power  of  infection. 
Diphtheria,  on  the  other  hand,  is,  as  we  have  pointed  out, 
capable  of  lengthened  survival  outside  the  body,  particularly 

1  Public  Health,  vol.  x.,  No.  4,  p.  130  (1898). 

2  Fliigge,  Grundriss  der  Hygiene,  1897. 

3  Zeitschrift  fur  Hygiene,  vols.  xxiv.-xxvi. 


BACTERIA    IN   THE   AIR  109 

when  surrounded  with  dust.  The  question  of  their  power 
of  resisting  long  drying  is  an  unsettled  point.  The  power  of 
surviving  a  drying  process  is,  according  to  Germano,  pos- 
sessed by  the  streptococcus.  This  is  not  the  case  with 
cholera  or  plague.  Dr.  Germano  classifies  bacteria,  as  a 
result  of  his  researches,  into  three  groups :  first,  those  like 
plague,  typhoid,  and  cholera,  which  cannot  survive  drying 
for  more  than  a  few  hours;  second,  those  like  the  bacilli  of 
diphtheria,  and  streptococci,  which  can  withstand  it  for  a 
longer  period ;  thirdly,  those  like  tubercle,  which  can  very 
readily  resist  drying  for  months  and  yet  retain  their  virul- 
ence. It  will  be  obvious  that  from  these  data  it  is  inferred 
that  Groups  I  and  2  are  rarely  conveyed  by  the  air,  whereas 
Group  3  is  frequently  so  conveyed.  Miquel  has  recently 
demonstrated  that  soil  bacteria  or  their  spores  can  remain 
alive  in  hermetically  sealed  tubes  for  as  long  a  time  as 
sixteen  years.  Even  at  the  end  of  that  period  the  soil 
inoculated  into  a  guinea-pig  produced  tetanus.1 

The  presence  of  pathogenic  bacteria  in  the  air  is,  of 
course,  a  much  rarer  contamination  than  the  ordinary  sap- 
rophytes. Tubercle  has  been  not  infrequently  isolated  from 
dry  dust  in  consumption  hospitals,  and  in  exit  ventilating 
shafts  at  Brompton  the  bacillus  has  been  found.  From 
dried  sputum  it  has,  of  course,  been  many  times  isolated, 
even  after  months  of  desiccation.  M.  Lalesque  failed  to 
isolate  it  from  the  dry  soil  surrounding  some  garden  seats 
in  a  locality  frequented  by  phthisical  patients.  The  writer 
also  failed  to  isolate  it  from  the  same  soil.  But  a  very  large 
mass  of  experimental  evidence  attests  the  fact  that  the  air 
in  proximity  to  dried  tubercular  sputum  or  discharges  may 
contain  the  specific  bacillus  of  the  disease.  Diphtheria  in 
the  same  way,  but  in  a  lesser  degree,  may  be  isolated  from 
the  air,  and  from  the  nasal  mucous  membrane  of  nurses, 
attendants,  and  patients  in  a  ward  set  apart  for  the  treat- 

:  Annales  de  Micrographie. 


HO  BACTERIA 

ment  of  the  disease,  Delalivesse,  examining  the  air  of  wards 
at  Lille,  found  that  the  contained  bacteria  varied  more  or 
less  directly  with  the  amount  of  floating  matter,  and  de- 
pended also  upon  the  vibration  set  up  by  persons  passing 
through  the  ward  and  the  heavy  traffic  in  granite-paved 
streets  adjoining.  Bacillus  coli,  staphylococci,  and  strepto- 
cocci, as  well  as  B.  tuberculosis,  were  isolated  by  this  observer. 
Some  new  light  has  been  thrown  upon  the  subject  of 
pathogenic  organisms  in  air  by  Neisser  in  his  investigations 
concerning  the  amount  and  rate  of  air-currents  necessary  to 
convey  certain  species  through  the  atmosphere.  He  states 
that  the  bacteria  causing  diphtheria,  typhoid  fever,  plague, 
cholera,  and  pneumonia,  and  possibly  the  common  Strepto- 
coccus pyogenes,  are  incapable  of  being  carried  by  the  mol- 
ecules of  atmospheric  dust  which  the  ordinary  insensible 
currents  of  air  can  support,  whilst  Bacillus  anthracis,  B. 
pyocyaneus,  and  the  bacillus  of  tubercle  are  capable  of  being 
aerially  conveyed0  This  work  will  require  further  confirm- 
ation, but  if  its  truth  be  established,  it  proves  that  attempted 
aerial  disinfection  of  the  first  group  of  diseases  is  useless. 


CHAPTER  IV 

BACTERIA  AND  FERMENTATION 


IT  was  Pasteur  who  in  1857  &rst  propounded  the  true  cause 
and  process  of  fermentation.  The  breaking  down  of 
sugar  into  alcohol  and  carbonic  acid  gas  had  been  known, 
of  course,  for  a  long  period.  Since  the  time  of  Spallanzani 
(1776)  the  putrefactive  changes  in  liquids  and  organic  matter 
had  been  prevented  by  boiling  and  subsequently  sealing  the 
flask  or  vessel  containing  the  fluid.  Moreover,  this  success- 
ful preventive  practice  had  been  in  some  measure  correctly 
interpreted  as  due  to  the  exclusion  of  the  atmosphere,  but 
wrongly  credited  to  the  exclusion  of  the  oxygen  of  the  air. 
It  was  not  until  the  beginning  of  the  present  century  that 
authorities  modified  their  view  and  declared  in  favour  of 
yeast  cells  as  the  agents  in  the  production  of  fermentation. 
That  this  process  was  due  to  oxygen  per  se  was  disproved 
by  Schwann,  who  showed  that  so  long  as  the  oxygen  ad- 
mitted to  the  flask  of  fermentative  fluid  was  sterilised  no 
fermentation  occurred.  It  was  thus  obvious  that  it  was  not 
the  atmosphere  or  the  oxygen  of  the  atmosphere,  but  some 
fermenting  agent  borne  into  the  flask  by  the  admission  of 
unsterilised  air.  It  was  but  a  step  to  further  establish  this 
hypothesis  by  adding  unsterilised  air  plus  some  antiseptic 
substance  which  would  destroy  the  fermenting  agent. 
Arsenic  was  found  by  Schwann  to  have  this  germicidal 
faculty.  Hence  Schwann  supported  Latour's  theory  that 
fermentation  was  due  to  something  borne  in  by  the  air,  and 


112  BACTERIA 

that  this  something  was  yeast.  Passing  over  a  number  of 
counter-experiments  of  Helmholtz  and  others,  we  come  to 
the  work  of  Liebig.  (  He  viewed  the  transformation  of  sugar 
into  alcohol  and  carbonic  acid  gas  simply  and  solely  as  a 
non-vital  chemical  process,  depending  upon  the  dead  yeast 
communicating  its  own  decomposition  to  surrounding  ele- 
ments in  contact  with  it.  ) 

Liebig  insisted  that  all  albuminoid  bodies  were  unstable, 
and  if  left  to  themselves  would  fall  to  pieces — i.  e.,  ferment 
— without  the  aid  of  living  organisms,  or  any  initiative  force 
greater  than  dead  yeast  cells.  It  was  at  this  juncture  that 
Pasteur  intervened  to  dispel  the  obscurities  and  contradic- 
tory theories  which  had  been  propounded. 

As  in  all  the  conclusions  arrived  at  by  Pasteur,  so  in  those 
relating  to  fermentation,  there  were  a  number  of  different 
experiments  which  were  performed  by  him  to  elucidate  the 
same  point.  We  will  choose  one  of  many  in  relation  to 
fermentation.  If  a  sugary  solution  of  carbonate  of  lime  is 
left  to  itself,  after  a  time  it  begins  to  effervesce,  carbonic 
acid  is  evolved,  and  lactic  acid  is  formed ;  and  this  latter 
decomposes  the  carbonate  of  lime  to  form  lactate  of  lime. 
This  lactic  acid  is  formed,  so  to  speak,  at  the  expense  of 
the  sugar,  which  little  by  little  disappears.  Pasteur  demon- 
strated the  cause  of  this  transformation  of  sugar  into  lactic 
acid  to  be  a  thin  layer  of  organic  matter  consisting  of  ex- 
tremely small  moving  organisms.  If  these  be  withheld  or 
destroyed  in  the  fermenting  fluid,  fermentation  will  cease. 
If  a  trace  of  this  grey  material  be  introduced  into  sterile 
milk  or  sterile  solution  of  sugar,  the  same  process  is  set  up, 
and  lactic  acid  fermentation  occurs. 

Pasteur  examined  the  elements  of  this  organic  layer  by 
aid  of  the  microscope,  and  found  it  to  consist  of  small  short 
rods  of  protoplasm  quite  distinct  from  the  yeast  cells  which 
previous  investigators  had  detected  in  alcoholic  fermenta- 
tion. One  series  of  experiments  was  accomplished  with 


BACTERIA   AND  FERMENTATION  113 

yeast  cells  and  these  bacteria,  a  second  series  with  living 
yeast  cells  only,  a  third  series  with  bacteria  only,  and  the 
conclusions  which  Pasteur  arrived  at  as  the  result  of  these 
labours  were  as  follows : 

"  As  for  the  interpretation  of  the  group  of  new  facts  which 
I  have  met  with  in  the  course  of  these  researches,  I  am  confident 
that  whoever  shall  judge  them  with  impartiality  will  recognise 
that  the  alcoholic  fermentation  is  an  act  correlated  to  the  life  and 
to  the  organisation  of  these  corpuscles,  and  not  to  their  death  or 
their  putrefaction,  any  more  than  it  will  appear  as  a  case  of  contact 
action  in  which  the  transformation  of  the  sugar  is  accomplished 
in  the  presence  of  the  ferment  without  the  latter  giving  or  taking 
anything  from  it." 

Pasteur  occupied  six  years  (1857-1863)  with  further  eluci- 
dation of  his  wonderful  discovery  of  the  potency  of  these 
hitherto  unrecognised  agents,  and  the  establishment  of  the 
fact  that  "  organic  liquids  do  not  alter  until  a  living  germ 
is  introduced  into  them,  and  living  germs  exist  every- 
where." 

It  must  not  be  supposed  that  to  Pasteur  is  due  the  whole 
credit  of  the  knowledge  acquired  respecting  the  cause  of 
fermentation.  He  did  not  first  discover  these  living  organ- 
isms; he  did  not  first  study  them  and  describe  them;  he 
was  not  even  the  first  to  suggest  that  they  were  the  cause 
of  the  processes  of  fermentation  or  disease.  But,  neverthe- 
less, it  was  Pasteur  who  "  first  placed  the  subject  upon  a 
firm  foundation  by  proving  with  rigid  experiment  some  of 
the  suggestions  made  by  others."  Thus  it  has  ever  been 
in  the  times  of  new  learning  and  discovery:  many  contribu- 
tors have  added  their  quota  to  the  mass  of  knowledge,  even 
though  one  man  appearing  at  the  right  moment  has  drawn 
the  conclusions  and  proved  the  theory  to  be  fact. 

In  order  that  no  confusion  may  arise  in  the  mind  of  the 
reader,  we  may  here  say  that,  although  fermentation  is 


114  BACTERIA 

always  due  to  a  living  agent,  as  proved  by  Pasteur,  the 
process  is  conveniently  divided  into  two  kinds.1  (i)  When 
the  action  is  direct,  and  the  chemical  changes  involved  in 
the  process  occur  only  in  the  presence  of  the  cell,  the  latter 
is  spoken  of  as  an  organised  ferment ;  (2)  when  the  action  is 
indirect,  and  the  changes  are  the  result  of  the  presence  of  a 
soluble  material  secreted  by  the  cell,  acting  apart  from  the 
cell,  this  soluble  substance  is  termed  an  unorganised  soluble 
ferment ,  or  enzyme.  The  organised  ferments  are  bacteria  or 
vegetable  cells  allied  to  the  bacteria;  the  unorganised  fer- 
ments, or  enzymes,  are  ferments  found  in  the  secretions  of 
specialised  cells  of  the  higher  plants  and  animals.  With  the 
former  this  book  deals  in  an  elementary  fashion ;  with  the 
latter  we  have  little  concern.  It  will  be  sufficient  to  illus- 
trate the  enzymes  by  a  few  of  the  more  familiar  examples. 
They  form,  for  example,  the  digestive  agents  in  human  as- 
similation. This  function  is  performed,  in  some  cases,  by  the 
enzyme  combining  with  the  substance  on  which  it  is  acting 
and  then  by  decomposition  yielding  the  new  "  digested  " 
substance  and  regenerating  the  enzyme ;  in  other  cases,  the 
enzyme,  by  its  molecular  movement,  sets  up  molecular 
movement  in  the  substance  it  is  digesting,  and  thus  changes 
its  condition.  These  digestive  enzymes  are  as  follows :  in 
the  saliva,  ptyalin,  which  changes  starch  into  sugar;  in  the 
gastric  juice  of  the  stomach,  pepsin,  which  digests  the  pro- 
teids  of  the  food  and  changes  them  into  absorptive  pep- 
tones; the  pancreatic  ferments,  amylopsin,  trypsin,  and 
steapsin,  capable  of  attacking  all  three  classes  of  food  stuffs; 
and  the  intestinal  ferments,  which  have  not  yet  been  sepa- 
rated in  purer  condition.  In  addition  to  these,  there  are 
ferments  in  bitter  almonds,  mustard,  etc.  Concerning 
these  unorganised  ferments  we  have  nothing  further  to  say. 
Perhaps  the  commonest  of  them  all  is  diastase,  which  occurs 
in  malt,  and  to  which  some  reference  will  be  made  later. 

1  E.  A.  Schafer,  F.R.S.,   Text-book  on  Physiology,  vol.  i.,  p.  312. 


BACTERIA   AND  FERMENTATION  115 

Its  function  is  to  convert  the  starch  which  occurs  in  barley 
into  sugar.  These  unorganised  ferments  act  most  rapidly 
at  about  75°  C.  (167°  F.).1 

We  may  now  return  to  the  work  of  Pasteur  and  the  ques- 
tion of  organised  ferments.  Let  us  preface  further  remark 
with  an  axiom  with  which  Professor  Frankland  sums  up  the 
vitalistic  theory  of  fermentation,  which  was  supported  by 
the  researches  of  Pasteur:  4< No  fermentation  without  organ- 
isms, in  every  fermentation  a  particular  organism. ' '  From 
these  words  we  gather  that  there  is  no  one  particular  organ- 
ism or  vegetable  cell  to  be  designated  the  micro-organism  of 
fermentation,  but  that  there  are  a  number  of  fermentations 
each  started  by  some  specific  form  of  agent.  It  is  true  that 
the  chemical  changes  induced  by  organised  ferments  de- 
pend on  the  life  processes  of  micro-organisms  which  feed 
upon  the  sugar  or  other  substance  in  solution,  and  excrete 
the  product  of  the  fermentation.  Fermentation  nearly 
always  consists  of  a  process  of  breaking  down  of  complex 
bodies,  like  sugar,  into  simpler  ones,  like  alcohol  and  car- 
bonic acid.  Of  such  fermentation  we  may  mention  at  least 
five  :  the  alcoholic,  by  which  alcohol  is  produced ;  the  acetous, 
by  which  wine  absorbs  oxygen  from  the  air  and  becomes 
vinegar;  the  lactic,  which  sours  milk;  the  butyric,  which  out 
of  various  sugars  and  organic  acids  produces  butyric  acid ; 
and  ammoniac al,  which  is  the  putrefactive  breaking  down 
of  compounds  of  nitrogen  into  ammonia.  We  have  already 
referred  at  some  length  to  this  process  when  considering 
denitrifying  organisms  in  the  soil. 

1  The  unorganised  ferments  are  frequently  otherwise  classified  than  as  above, 
not  according  to  the  locality,  but  according  to  the  function.  The  chief  are 
these  : — amylolytic,  those  which  change  starch  and  glycogen  (amyloses)  into 
sugars,  e.  g.,  ptyalin,  diastase,  amylopsin  ;  proteolytic,  those  which  change 
proteids  into  proteosis  and  peptones,  e.  g.,  trypsin,  pepsin  ;  inversive,  those 
which  change  maltose,  sucrose,  and  lactose  into  glucose,  e.g.,  invertin  ;  coag-u- 
lative,  those  which  change  soluble  proteids  into  insoluble,  e.  g.,  rennet; 
steatolytie,  those  which  split  up  fats  into  fatty  acids  and  glycerine,  e.  g.,  steapsin. 


Il6  BACTERIA 

^There  are  four  chief  conditions  common  to  all  these  five 
kinds  of  organised  fermentation.     They  are  as  follows : — 

1.  The  presence  of  the  special  living  agent  or  organism  of 
the  particular  fermentation  under  consideration.     This,  as 
Pasteur  pointed  out,  differs  in  each  case. 

2.  A  sufficiency  of  pabulum  (nutriment)  and  moisture  to 
favour  the  growth  of  the  micro-organism. 

3.  A  temperature    at   or  about    blood-heat  (35-38°   C, 

98.5°  F.). 

4.  The  absence  from  the  solution  or  substance  of  any 
obnoxious  or  inimical  substances  which  would  destroy  or 
retard  the  action  of  the  living  organism  and  agent.     Many 
of  the  products  of  fermentation  are  themselves  antiseptics, 
as   in   the   case   of  alcohol;    hence   alcoholic   fermentation 
always  arrests  itself  at  a  certain  point. 

We  are  now  in  a  position  to  consider  particular  ferment- 
ations and  their  causal  micro-organisms.  These  latter  are  of 
various  kinds,  belonging,  according  to  botanical  classifica- 
tion, to  various  different  subdivisions  of  the  non-flowering 
portion  of  the  vegetable  kingdom.  A  large  part  of  ferment- 
ation is  based  upon  the  growth  of  a  class  of  microscopic 
plants  termed  yeasts.  These  differ  from  the  bacteria  in  but 
few  particulars,  mainly  in  their  method  of  reproduction  by 
budding  (instead  of  dividing  or  sporulating,  like  the  bac- 
teria). Their  chemical  action  is  closely  allied  to  that  of  the 
bacteria.  Secondly,  there  are  special  fermentations  and 
modifications  of  yeast  fermentation  due  to  bacteria.  Thirdly, 
a  group  of  somewhat  more  highly  specialised  vegetable  cells, 
known  as  moulds,  make  a  perceptible  contribution  in  this 
direction.  According  to  Hansen,  these  latter,  so  far  as 
they  are  really  alcoholic  ferments,  induce  fermentation,  not 
only  in  solutions  of  dextrose  and  invert  sugar,  but  also  in 
solutions  of  maltose.  Mucor  racemosus  is  the  only  member 
that  is  capable  of  inverting  a  cane-sugar  solution  ;  M.  erectus 
is  the  most  active  fermenter,  yielding  eight  per  cent,  by 


BACTERIA   AND  FERMENTATION  1 1/ 

volume  of  alcohol  in  ordinary  beer  wort.  Each  of  these 
will  be  referred  to  as  they  occur  in  considering  the  five  im- 
portant fermentations  already  mentioned. 

The  general  microscopic  appearance  of  yeast  cells  may  be 
shortly  stated  as  follows :  they  are  round  or  oval  cells,  and 
by  budding  become  daughter  yeasts.  Each  consists  of  a 
membrane  and  clear  homogeneous  contents.  As  they  per- 
form their  function  of  fermentation,  vacuoles,  fat-globules, 


cO 


e> 


Cb    CD 


SACCHAROMYCES  CEREVISL*: 

and  other  granules  make  their  appearance  in  the  enclosed 
plasma.  As  in  many  vegetable  cells  a  nucleus  was  detected 
by  Schmitz  by  means  of  special  methods  of  staining,  Han- 
sen  has  found  the  nucleus  in  old  yeast  cells  from  "  films  " 
without  any  special  staining. 

I.   Alcoholic  Fermentation. 

Cause,  yeast  ;  medium,  sugar  solutions  ;  result,  alcohol  and  carbonic  acid. 

It  was  Caignard-Latour  who  first  demonstrated  that  yeast 
cells,  by  their  growth  and  multiplication,  set  up  a  chemical 
change  in  sugar  solutions  which  resulted  in  the  transference 
of  the  oxygen  from  the  hydrogen  in  the  sugar  compound  to 
the  carbon  atoms,  that  is  to  say,  in  the  evolution  of  carbonic 
acid  gas  and  the  production,  as  a  result,  of  alcohol.  If  we 
were  to  express  this  in  a  chemical  formula,  it  would  read  as 
follows : 


Il8  BACTERIA 

C6  Hj  2  O6  (plus  the  yeast)  =  2  C2  H6  O  +  2  CO2. 

A  natural  sugar,  like  grape-sugar,  present  in  the  fruit  of 
the  vine,  is  thus  fermented.  The  alcohol  remains  in  the 
liquid ;  the  carbonic  acid  escapes  as  bubbles  of  gas  into  the 
surrounding  air.  It  is  thus  that  brandy  and  wines  are  made. 
If  we  go  a  step  further  back,  to  cane-sugar  (which  possesses 
the  same  elements  as  grape-sugar,  but  in  different  propor- 
tions), dissolve  it  in  water,  and  mix  it  with  yeast,  we  get 
exactly  the  same  result,  except  that  the  first  stage  of  the 
fermentation  would  be  the  changing  of  the  cane-sugar  into 
grape-sugar,  which  is  accomplished  by  a  soluble  ferment 
secreted  by  the  yeast  cells  themselves.  If  now  we  go  yet 
one  step  further  back,  to  starch,  the  same  sort  of  action 
occurs.  When  starch  is  boiled  with  a  dilute  acid  it  is 
changed  into  a  gum-like  substance  named  dextrin,  and  sub- 
sequently into  a  sugar  named  maltose,  which  latter,  when 
mixed  with  these  living  yeast  cells,  is  fermented,  and  results 
in  the  evolution  of  carbonic  acid  gas  and  the  production  of 
alcohol.  In  the  manufacture  of  fermented  drinks  from 
cereal  grains  containing  starch  there  is  therefore  a  double 
chemical  process :  first  the  change  of  starch  into  sugar  by 
means  of  conversion?  and  secondly  the  change  of  the  sugar 
into  alcohol  and  carbonic  acid  gas  by  the  process  of  fer- 
mentation, an  organic  change  brought  about  by  the  living 
yeast  cells. 

In  all  these  three  forms  of  alcoholic  fermentation  the 
principal  features  are  the  same,  viz.,  the  sugar  disappears; 
the  carbonic  acid  gas  escapes  into  the  air;  the  alcohol  re- 
mains behind.  Though  it  is  true  that  the  sugar  disappears, 
it  would  be  truer  still  to  say  that  it  reappears  as  alcohol. 
Sugar  and  alcohol  are  built  up  of  precisely  the  same  ele- 
ments: carbon,  hydrogen,  and  oxygen.  They  differ  from 

1  A  chemical  change  obtained  by  the  action  of  sulphuric  or  some  other  acid, 
or  by  the  influence  of  diastase. 


BACTERIA    AND  FERMENTATION  119 

each  other  in  the  proportion  of  these  elements.  It  is 
obvious,  therefore,  that  fermentation  is  really  only  a  change 
of  position,  a  breaking  down  of  one  compound  into  two 
simpler  compounds.  This  redistribution  of  the  molecules 
of  the  compound  results  in  the  production  of  some  heat. 
Thus  we  must  add  heat  to  the  results  of  the  work  of  the 
yeasts. 

When  alcohol  is  pure  and  contains  no  water  it  is  termed 
absolute  alcohol.  If,  however,  it  is  mixed  with  16  per  cent, 
of  water,  it  is  called  rectified  spirit,  and  when  mixed  with 
more  than  half  its  volume  of  water  (56.8  per  cent.)  it  is 
known  as  proof  spirit. 

We  shall  have  to  consider  elsewhere  a  remarkable  faculty 
which  some  bacteria  possess  of  producing  products  inimical 
to  their  own  growth.  In  some  degree  this  is  true  of  the 
yeasts,  for  when  they  have  set  up  fermentation  in  a  saccha- 
rine fluid  there  comes  a  time  when  the  presence  of  the  re- 
sulting alcohol -is  injurious  to  further  action  on  their  part. 
It  has  become  indeed  a  poison,  and,  as  we  have  already 
mentioned,  a  necessary  condition  for  the  action  of  a  ferment 
is  the  absence  of  poisonous  substances.  This  limit  of  fer- 
mentation is  reached  when  the  fermenting  fluid  contains  13 
or  14  per  cent,  of  alcohol. 

Having  discussed  shortly  the  "  medium  "  and  the  results, 
we  may  now  turn  to  the  bacteriology  of  the  matter,  and 
enumerate  some  of  the  chief  forms  of  the  yeast  plant.  Pro- 
fessor Crookshank 1  gives  more  than  a  score  of  different 
members  of  this  family  of  Saccharomycetes.  Before  dwelling 
upon  some  of  the  chief  of  these,  it  will  be  desirable  to  con- 
sider a  number  of  properties  common  to  the  genus. 

The  yeast  cell  is  a  round  or  oval  body  of  the  nature  of  a 
fungus,  composed  of  granular  protoplasm  surrounded  by  a 
definite  envelope,  or  capsule.  It  reproduces  itself  by  bud- 
ding, or,  as  it  is  sometimes  termed,  gemmation.  At  one  end 

1  Bacteriology  and  Infective  Diseases,  Appendix. 


I2O  BACTERIA 

of  the  cell  a  slight  swelling  or  protuberance  appears,  which 
slowly  enlarges.  Ultimately  there  is  a  constriction,  and  the 
bud  becomes  partly  and  at  last  completely  separated  from 
the  parent  cell.  In  many  cases  the  capsules  of  the  daugh- 
ter cell  and  the  parent  cell  adhere,  thus  forming  a  chain  of 
budding  cells.  The  character  of  the  cell  and  its  method  of 
reproduction  do  not  depend  merely  upon  the  particular 
species  alone,  but  are  also  dependent  upon  external  circum- 
stances. There  are  differences  in  the  behaviour  of  species 
towards  different  media  at  various  temperatures,  towards  the 


O 


ASCOSPORE  FORMATION 

carbohydrates  (especially  maltose),  and  in  the  chemical 
changes  which  they  bring  about  in  nutrient  liquids.  In 
connection  with  this  Professor  Hansen  has  pointed  out  that, 
whilst  some  species  can  be  made  use  of  in  fermentation 
industries,  others  cannot,  and  some  even  produce  diseases 
in  beer.1 

One  of  the  most  remarkable  evidences  of  the  adaptability 
of  the  yeasts  to  their  surroundings  and  a  specific  character- 
istic occurs  in  what  is  sometimes  called  ascospore  formation. 
If  a  yeast  cell  finds  itself  lacking  nourishment  or  in  an  un- 
favourable medium,  it  reproduces  itself  not  by  budding,  but 
by  forming  spores  out  of  its  own  intrinsic  substance,  and 
within  its  own  capsule.  To  obtain  this  kind  of  spore  form- 
ation Hansen  used  some  gypsum  blocks  as  medium  on  which 

1  E.  C.  Hansen,  Studies  in  Fermentation  (Copenhagen),  p.  98. 


BACTERIA   AND  FERMENTATION 


121 


to  grow  his  yeast  cells.  Well-baked  plaster  of  Paris  is 
mixed  with  distilled  water,  and  made  into  a  liquid  paste. 
Small  moulds  are  made  by  pouring  this  paste  into  card- 
board dishes,  where  it  hardens  again.  The  mould  is  steril- 
ised by  heat,  and  a  small  portion  of  yeast  is  placed  on  its 
upper  surface,  and  then  the  whole  is  floated  in  a  small  ves- 
sel of  water  and  covered  with  a  bell-jar.  Under  these  con- 
ditions of  limited  pabulum  the  cell  undergoes  the  following 
changes :  it  increases  in  size,  loses  much  of  its  granularity, 
and  becomes  homogeneous,  and  about  thirty  hours  after 


GYPSUM  BLOCK 

being  sown  on  the  gypsum  there  appear  several  refractile 
cells  inside  the  parent  cell.  These  are  the  ascospores.  In 
addition  to  the  gypsum,  it  is  necessary  to  have  a  plentiful 
supply  of  oxygen,  some  moisture  (gained  from  the  vessel  of 
water  in  which  the  gypsum  floats),  a  certain  temperature, 
and  a  young  condition  of  the  protoplasm  of  the  parent  yeast 
cells.  Hansen  found  that  the  lowest  temperature  at  which 
these  ascospores  were  produced  was  .5-3°  C.,  and  at  the 
other  extreme  up  to  37°  C.,  which  is  blood-heat.  The 
rapidity  of  formation  also  varies  with  the  temperature,  the 
favourable  degree  of  warmth  being  about  22-25°  C. 

Hansen  pointed  out  that  it  was  possible  by  means  of 
sporulation  to  differentiate  species  of  yeasts.  For  it  hap- 
pens that  different  species  show  slight  differences  in  spore 
formation,  e.  g.  : 


122  BACTERIA 

(a)  The  spores  of  Saccharomyces  cerevisia  expand  during 
the  first  stage  of  germination,  and  produce  partition  walls, 
making  a  compound  cell  with  several  chambers.     Budding 
can  occur  at  any  point  on  the  surface  of  the  swollen  spores. 
To  this  group  belong  6".  pastorianus  and  »S.  ellipsoideus. 

(b)  The  spores  of  Saccharomyces  Ludwigii  fuse  in  the  first 
stage,  and  afterwards  grow  out  into  a  promycelium,  which 
produces  yeast  cells. 

(c)  The  spores  of  Saccharomyces  anomalus  are  different  in 
shape  from  the  others  in  that  they  possess  a  projecting  rim 
round  the  base. 

Another  point  in  the  cultivation  of  yeasts  has  been  eluci- 
dated by  a  number  of  workers,  chief  among  whom  perhaps 
is  Hansen,  namely,  methods  of  obtaining  pure  cultures.  We 
know,  generally  speaking,  what  this  term  means,  and  there 
is  no  difference  in  its  meaning  here  to  what  is  understood  as 
its  meaning  with  regard  to  bacteria.  There  is,  however, 
some  difference  in  the  mode  of  securing  it.  It  is  only  by 
starting  with  one  individual  cell  that  we  can  hope  to  secure 
a  pure  culture  of  yeasts.  For  the  study  of  the  morphology 
of  yeasts  under  the  microscope  the  problem  was  not  a  diffi- 
cult one.  It  was  comparatively  easy  to  keep  out  foreign 
germs  from  a  cover-glass  preparation  enough  to  perceive 
germination  of  spores  and  growth  of  mycelium.  But  when 
we  require  pure  cultures  for  various  physiological  purposes, 
then  a  different  standard  and  method  are  necessary. 

Pasteur  and  Cohn  adopted  a  practice  based  upon  the  fact 
that  when  organisms  find  themselves  in  a  favourable  medium 
they  multiply  to  the  exclusion  of  others  to  which  the  medium 
is  less  favourable.  Hence  if  an  impure  mixture  be  placed 
under  such  circumstances  there  comes  a  time  when  those 
organisms  for  which  the  circumstances  are  favourable  mul- 
tiply to  such  an  extent  that  they  form  an  almost  pure  cult- 
ure. The  method  is  open  to  fallacy,  and  will  rarely  result 
in  a  really  pure  culture;  and  even  if  that  be  secured,  it  is 


t 


YEAST  (SACCHAROMYCES  CEREVISI/E) 


X  1000 


ASCOSPORE  FORMATION  IN  YEAST 

(The  capsule  of  the  parent  cell  around  the  spores 

is  invisible) 

X   1000 


MITROGEN  FIXING  BACTERIA  FROM 
ROOTLET-NODULES  (SUBCULTURE) 

X  1000 


.  ' 

\f. 


BACILLUS  OF  TETANUS 

(From  broth  culture,  showing  spore  formation) 
X   1000 

By  permission  of  the  Scientific  Press^  Limited 


BACTERIA   AND  FERMENTATION  1 23 

quite  possible  that  it  will  be  to  the  exclusion  of  the  desired 
culture.  Hansen  has  devised  a  much  improved  process  for 
securing  a  pure  culture  of  yeast  which  depends  upon  dilu- 
tion. We  believe  Lister  was  one  of  the  first  who,  in  the 
seventies,  introduced  some  such  plan  as  this.  Hansen  em- 
ployed dilution  with  water  in  the  following  manner: 

Yeast  is  diluted  with  a  certain  amount  of  sterilised  water. 
A  drop  is  carefully  examined  under  the  microscope,  a  single 
cell  of  yeast  is  taken,  and  a  cultivation  made  upon  wort. 
When  it  has  grown  abundantly  a  quantity  of  sterilised  water 
is  added.  From  this,  again,  a  single  drop  is  taken  and 
added,  to,  say  20  cc.  of  sterilised  water  in  a  fresh  flask.  This 
flask  will  contain  we  will  suppose  ten  cells.  It  is  now  vigor- 
ously shaken,  and  the  contents  are  divided  into  twenty  por- 
tions of  I  cc.  each,  and  added  to  twenty  tubes  of  sterilised 
water.  It  is  highly  probable  that  half  of  those  tubes  have 
received  one  cell  each.  In  the  course  of  a  few  days  it  can 
be  seen  how  far  a  culture  is  pure.  If  only  one  colony  is 
present,  the  culture  is  a  pure  one,  and  as  this  grows  we 
obtain  an  absolutely  pure  culture  in  necessary  quantity. 
Even  when  the  gelatine-plate  method  is  used  it  is  desirable 
to  start  with  a  single  cell  (Hansen).  The  advantage  of 
Hansen's  yeast  method  over  Koch's  bacterial-plate  method 
is  that  it  has  a  certain  definite  starting-point.  This  is 
obviously  impossible  when  dealing  with  such  microscopic 
particles  as  the  bacteria  proper. 

A  third  matter  in  the  differentiation  of  yeast  species  is 
the  question  of  films.  Hansen  set  to  work,  after  having 
obtained  pure  cultures  and  ascospores,  to  examine  films  ap- 
pearing on  the  surface  of  liquids  undergoing  fermentation. 
The  object  of  this  was  to  ascertain  whether  all  yeasts  pro- 
duced the  same  mycelial  growth  on  the  surface  of  the 
fermenting  fluid.  To  produce  these  films  the  process  is  as 
follows :  Drop  on  to  the  surface  of  sterilised  wort  in  a  flask 
a  very  small  quantity  of  a  pure  culture  of  yeast ;  secure  the 


124  BACTERIA 

flask  from  movement,  and  protect  it,  not  from  air,  which  is 
necessary,  but  from  falling  particles  in  the  air.  In  a  short 
time  small  colonies  appear,  which  coalesce  and  form  patches, 
then  a  film  or  membrane  which  covers  the  liquid  and  attaches 
itself  to  the  sides  of  the  flask.  By  the  differences  in  the 
films  and  the  temperatures  at  which  they  form  it  is  possible 
to  obtain  something  of  a  basis  for  classification.  The  further 
advances  in  a  yeast  culture  and  in  our  knowledge  of  the  agen- 
cies of  fermentation  have,  however,  tended  to  show  that  no 
strict  dividing  lines  can  be  drawn.  Hansen's  researches  have, 
notwithstanding,  been  of  the  greatest  moment  to  the  whole 
industry  of  fermentation.  What  has  been  found  true  in 
bacteriology  has  also  been  demonstrated  in  fermentation, 
namely,  that  though  many  yeasts  differ  but  little  in  struct- 
ure and  behaviour,  they  may  produce  very  different  products 
and  possess  very  different  properties.  Industrial  cultivation 
of  these  finer  differences  in  fermentative  action  has  to  a  large 
extent  revolutionised  the  brewing  industry. 

The  formation  of  films  is  not  a  peculiarity  of  certain 
species,  but  must  be  regarded  as  a  phenomenon  occurring 
somewhat  commonly  amongst  yeasts.  The  requisites  are 
a  suitable  medium,  a  yeast  cell,  a  free,  still  surface,  direct 
access  of  air,  and  a  favourable  temperature.  The  wort  loses 
colour,  and  becomes  pale  yellow.  Microscopic  differences 
soon  appear  between  the  sedimentary  yeast  and  the  film 
yeast  of  the  same  species,  the  latter  growing  out  into  long 
mycelial  forms,  the  character  of  which  depends  in  part  upon 
the  temperature.  This  often  varies  from  3°  to  38°  C. 

A  fourth  point  helpful  in  diagnosis  is  the  temperature 
which  proves  to  be  the  thermal  death-point.  Sac  char  omyces 
cerevisice  is  killed  by  an  exposure  to  54°  C.  for  five  minutes, 
and  62°  C.  kills  the  spores.  As  a  rule,  yeasts  can  resist  a 
considerably  higher  temperature  when  in  a  dry  state  than 
in  the  presence  of  moisture. 

Lastly,  yeasts  may  be  cultivated  on  solid  media.     Hansen 


BACTERIA    AND  FERMENTATION  12$ 

employed  wort-gelatine  (5  per  cent,  gelatine),  and  found 
that  at  25°  C.  in  a  fortnight  the  growths  which  develop 
show  such  microscopic  differences  as  to  aid  materially  in 
diagnosis.  Saccharomyces  ellipsoideus  L  exhibits  a  charac- 
teristic network  which  readily  distinguishes  it. 

There  is  one  other  point  to  which  reference  must  be 
made.  The  process  of  fermentation  may  be  set  up  by  a 
"  high  "  or  a  "  low"  yeast.  These  terms  apply  to  the 
temperature  at  which  the  process  commences.  "  High  " 
yeasts  rise  to  the  surface  as  the  action  proceeds,  accomplish 
their  work  rapidly,  and  at  a  comparatively  high  temper- 
ature, say  about  16°  C.  ;  "  low"  yeasts,  on  the  contrary, 
sink  in  the  fermenting  fluid,  act  slowly,  and  only  at  the  low 
temperature  of  4°  or  5°  C.  This  is  maintainable  by  floating 
ice  in  the  fluid.  Formerly  all  beer  was  made  by  the 
"  high  "  mode,  but  on  the  continent  of  Europe  "  low  " 
yeast  is  mostly  used,  while  the  "  high  "  is  in  vogue  in  Eng- 
land. This  latter  method  is  more  conducive  to  the  develop- 
ment of  extraneous  organisms,  and  therefore  risky  in  all  but 
well-ordered  brewing  establishments.  Whether  high  and 
low  yeasts  consist  of  one  or  several  species  is  not  known. 

Before  proceeding  to  mention  shortly  some  of  the  com- 
moner forms  of  yeast  we  must  again  emphasise  Hansen's 
method  of  analysis  in  separating  a  species.  The  shape, 
size,  and  appearance  of  cells  are  not  sufficient  for  different- 
iation, because  it  is  found  that  the  same  species  when  ex- 
posed to  different  external  conditions  can  occur  in  very 
different  forms.  Hence  Hansen  established  the  analytical 
method  of  observing  (i)  the  microscopic  appearance,  (2)  the 
formation  of  ascospores,  and  (3)  the  formation  of  films.  In 
addition,  the  temperature  limits,  cultivation  on  solid  media, 
and  behaviour  towards  carbohydrates,  are  characters  which 
aid  in  the  separation  of  yeasts.  By  basing  differentiation 
of  species  upon  these  features,  the  following  can  be  dis- 
tinguished : 


126 


BACTERIA 


SaccJiaromyces  Cerevisice.  Oval  or  ellipsoidal  cells ;  repro- 
duction by  budding;  ascospores,  rapidly  at  30°  C.,  slowly 
at  12°  C.,  not  formed  at  all  at  lower  temperatures;  film 
formation,  seven  to  ten  days  at  22°  C.  ;  an  active  alcoholic 
ferment,  producing  in  a  fortnight  in  beer  wort  from  4  to  6 
per  cent,  by  volume  of  alcohol  (Jorgensen).  This  species 
is  a  typical  English  "  high"  yeast,  possessing  the  power 
of  "  inverting  "  cane-sugar  previous  to  producing  alco- 
hol and  carbonic  acid.  It  is  said  to  have  no  action  on 
milk-sugar. 

Saccharomyces  Ellipsoideus  L  Round,  oval,  or  sausage- 
shaped  cells,  single  or  in  chains ;  ascospores  in  twenty-four 


>/'/ 


S.  ELLIPSOIDEUS 


S.  PASTORIANUS 


hours  at  25°  C.  (not  above  30°  C.,  not  below  4°  C.).  Grown 
on  the  surface  of  wort-gelatine,  a  network  is  produced  by 
which  they  can  be  recognised  (in  eight  to  twelve  days  at 
33°  C.).  At  13-15°  C.  a  characteristic  branching  mass  is 
produced.  It  is  an  alcoholic  ferment  as  active  as  5.  cere- 
visice.  S.  Ellipsoideus  II.  Round  and  oval,  rarely  elong- 
ated, a  widely  distributed  yeast,  causing  "  muddiness  "  in 
beer  and  a  bitter  taste.  It  is  essentially  a  "  low  "  yeast. 

Saccharomyces  Conglomeratus  is  a  round  cell,  often  united 
in  clusters,  and  occurring  in  rotting  grapes,  and  at  the  com- 
mencement of  fermentation. 


BACTERIA   AND  FERMENTATION 


Saccharomyces  Pastorianus  I.  Oval  or  club-shaped  cells, 
occurring  in  after-fermentation  of  wine,  etc.,  and  producing 
a  bitter  taste,  unpleasant  odour,  and  turbidity.  The  spores 
frequently  occur  in  the  air  of  breweries. 

5.  Pastor.  II.  Elongated  cells,  possessing  an  invertose 
ferment.  They  do  not,  like  5.  pastor  /.,  produce  disease  in 
beer. 

vS.  Pastor.  III.  Oval  or  elongated  cells,  producing  turbid- 
ity in  beer.  Grown  on  yeast-water  gelatine,  the  colonies 
show  after  sixteen  days  crenated  hairy  edges. 

Saccharomyces  Apiculatus.  Lemon-shaped  cells.  They 
give  rise  to  a  feeble  alcoholic  fermentation,  and  produce  two 
kinds  of  spores  —  round  and  oval  ;  they  appear  at  the  onset 
of  vinous  fermentation,  but  give  way  later  on  to  5.  cerevisice. 

Saccharomyces  My  coder  ma.  Oval  or  elliptical  cells,  often 
in  branching  chains.  They  form  the  so-called  "  mould  " 
on  fermented  liquids,  and  develop  on  the  surface  without 
exciting  fermentation.  When  forced  to  grow  submerged 
they  produce  a  little  alcohol. 

Saccharomyces  Exiguus.  Conical  cells,  appearing  in  the 
after-fermentation  of  beer. 

Saccharomyces  Pyriformis.  Oval  cells,  converting  sugary 
solutions  containing  ginger  into  ginger-beer. 

Saccharomyces  II  'lids  •,  Hansenii,  et  Aquifolii  produce  a 
small  percentage  of  alcohol. 

2.   Acetous  Fermentation. 

Cause,  Mycoderma  aceti  ;  medium,  wine  and  other  alcoholic  liquids  ;  result, 
the  formation  of  vinegar. 

If  alcohol  be  diluted  with  water,  and  the  specific  ferment 
mixed  with  it  and  exposed  to  the  air  at  22°  C.,  it  is  rapidly 
converted  into  vinegar.  The  change  is  accompanied  by  the 
absorption  of  oxygen,  one  atom  of  which  combines  with 
two  of  hydrogen  to  form  water,  and  a  substance  remains 


128  BACTERIA 

called  aldehyde,  further  oxidation   of  which  produces  the 
acetic  acid.     We  may  express  it  chemically  thus ; 

Alcohol.  Aldehyde.      Water. 

C2H6O  (+  oxygen  and  the  ferment)  =  C8H4O  -f  H2O. 

The  aldehyde  becomes  further  oxidised : 

C2H4O  +  O  =  C2H4O2  (acetic  acid). 

Now  this  method  of  simply  oxidising  alcohol  to  obtain 
acetic  acid  may  be  carried  out  chemically  without  any  fer- 
ment. If  slightly  diluted  alcohol  be  dropped  upon  platinum 
blacky  the  oxygen  condensed  in  that  substance  acts  with 
energy  upon  the  spirit,  and  union  readily  occurring,  acetic 
acid  results.  Here  the  whole  business  of  the  platinum 
sponge  is  to  persuade  the  oxygen  of  the  air  and  the  hydro- 
gen of  the  alcohol  to  unite.  In  the  ordinary  manufacture 
this  is  accomplished  by  the  vegetable  cells  of  Mycodenna 
aecti. 

There  are  two  chief  methods  adopted  in  the  commercial 
manufacture  of  vinegar,  both  of  which  depend  upon  the 
presence  of  the  Mycoderma.  The  method  in  vogue  at 
Orleans  when  Pasteur  (about  1862)  commenced  his  studies 
of  the  vinegar  organism  was  to  fill  vats  nearly  to  the  brim 
with  a  weak  mixture  of  vinegar  and  wine.  Where  the  pro- 
cess is  proceeding  the  surface  is  covered  with  a  fragile 
pellicle,  "  the  mother  of  vinegar,"  which  is  produced  by 
and  consists  of  certain  micro-organisms  whose  function  is 
to  convey  the  oxygen  of  the  air  to  the  liquor  in  the  vats, 
thus  oxidising  the  alcohol  into  vinegar.  This  oxidation 
may  be  carried  on  even  beyond  the  stage  of  acetic  acid 
(when  no  more  alcohol  remains  to  be  oxidised),  resulting  in 
carbonic  acid  gas,  which  escapes  into  the  air.  But  as  in  the 
alcoholic,  so  in  the  acetic,  fermentation,  there  comes  a  time 
when  the  presence  of  an  excess  of  the  acid  inhibits  the 
further  growth  of  the  organism.  This  point  is  approxim- 


BACTERIA    AND  FERMENTATION  129 

ately  when  the  acetic  acid  has  reached  a  percentage  as  high 
as  14.  But  if  the  acid  be  removed,  and  fresh  alcohol  added, 
the  process  recommences. 

The  second  method,  sometimes  called  by  the  Germans 
the  "  quick  vinegar  process,"  is  to  pour  the  weakened 
acohol  through  a  tall  cylinder  filled  with  wood-shavings, 
having  first  added  some  warm  vinegar  to  the  shavings. 
After  a  number  of  hours  the  resulting  fluid  is  charged  with 
acetic  acid.  What  has  occurred  ?  Liebig  maintained  that 
a  chemical  and  mechanical  change  had  brought  about  the 
change  from  the  alcohol  put  into  the  cylinder  and  the  vine- 
gar drawn  off  at  the  exit  tube.  It  was  reserved  for  Pasteur 
to  demonstrate  by  experiment  that  the  addition  of  the  warm 
vinegar  to  the  shavings  was  in  reality  an  addition  of  a  living 
micro-organism,  which,  forming  a  film  upon  the  shavings, 
became  "  the  mother  of  vinegar,"  and  oxidised  the  alcohol 
which  passed  over  it,  inducing  it  to  become  aldehyde  and 
then  acetic  acid. 

Mycoderma  Aceti  (described  by  Persoon  1822,  Kiitzing 
1837,  and  Pasteur  1864).  It  must  be  understood  that  this 
term  is  the  name  rather  of  a  family  than  an  individual. 
Pasteur  believed  it  to  be  a  specific  individual,  but  Hansen 
pointed  out  that  it  was  composed  of  two  distinctly  different 
species  (Bacterium  aceti  and  B.  pasteurianmri),  and  subse- 
quently other  investigators  have  added  members  to  the 
acetic  fermentation  group  of  which  M.  aceti  is  the  type. 

This  bacterium  is  made  up  of  small,  slightly  elongated 
cells,  with  a  transverse  diameter  of  2  or  3  /a,  sometimes 
united  in  short  chains  of  curved  rods.  They  frequently 
show  a  central  constriction,  are  motile,  and  produce  in  old 
cultures  involution  forms.  The  way  in  which  the  cells  act 
and  are  made  to  perform  their  function  is  as  follows:  A 
small  quantity,  taken  from  a  previous  pellicle,  is  sown  on 
the  surface  of  an  aqueous  liquid,  containing  2  per  cent,  of 
alcohol,  i  per  cent,  of  vinegar,  and  traces  of  alkaline  phos- 

9 


130  BACTERIA 

phates.  Very  rapidly  indeed  the  little  isolated  colonies 
spread,  and,  becoming  confluent,  form  a  membrane  or  pellicle 
over  the  whole  area  of  fluid.  When  the  surface  is  covered 
the  alcohol  acidifies  to  vinegar.  After  this  it  is  necessary 
to  add  each  day  small  quantities  of  alcohol.  When  the 
oxidation  is  completed  the  vinegar  is  drawn  off,  and  the 
membrane  is  collected  and  washed,  and  is  then  again  ready 
for  use.  It  ought  not  to  remain  long  out  of  fermenting 
liquid,  nor  ought  it  to  be  allowed  to  over-perform  its  func- 
tion, for  thus  having  oxidised  all  the  alcohol  it  will  com- 
mence oxidation  of  the  vinegar. 

In  wort-gelatine  Bacterium  pasteurianum  develops  round 
colonies  with  a  smooth  or  wavy  border,  whilst  B.  aceti  has  a 
tendency  towards  stellate  arrangement.  Spores  have  not 
been  observed,  and  from  a  morphological  point  of  view  the 
two  species  behave  alike.  Neither  produces  any  turbidity 
in  the  liquid  containing  them.  In  order  to  flourish,  B. 
aceti  requires  a  temperature  of  about  33°  C.  and  a  plentiful 
supply  of  oxygen.  In  a  cool  store  or  cellar  there  is,  there- 
fore, nothing  to  fear  from  B.  aceti.  Frankland  has  isolated 
a  Bacillus  ethaceticus,  which  is  a  fermentative  organism  pro- 
ducing ethyl-alcohol  and  acetic  acid.  By  oxidation  the 
ethyl-alcohol  may  be  converted  into  acetic  acid. 

3.   Lactic  Acid  Fermentation. 

Cause,  Bacillus  acidi  lactici  ;  medium,  milk-sugar,  cane-sugar,  glucose, 
dextrose,  etc.  ;  result,  lactic  acid. 

The  process  set  up  by  the  lactic  ferment  is  simply  a  de- 
composition, an  exact  division  of  one  molecule  of  sugar  into 
two  molecules  of  lactic  acid,  there  being  neither  oxidation 
nor  hydration.  The  conditions  under  which  the  ferment 
acts  are  very  similar  to  those  we  have  already  considered. 
There  is  frequently  carbonic  acid  gas  formed ;  there  is  a 
cessation  of  fermentation  when  the  medium  becomes  too 


BACTERIA    AND  FERMENTATION  131 

acid ;  there  is  the  same  method  of  starting  the  process  by 
inoculation  of  sour  milk  or  cheese  or  any  substance  contain- 
ing the  specific  bacillus.  It  is  probable  that  such  inoculated 
matter  will  contain  a  mixture  of  micro-organisms,  but  if  the 
lactic  bacillus  is  present,  it  will  grow  so  vigorously  and 
abundantly  that  the  fermentation  will  be  readily  set  up. 

TJie  Bacillus  Acidi  Lactici.     Rods  about  2  //,  long  and  4 /A 
wide,  occurring  singly  or  in  chains  and  threads.     It  is  non- 


CO     » 

B.  ACIDI  LACTICI 


motile.  Spore  formation  is  present,  the  spores  appearing 
irregularly  or  at  one  end  of  the  rod. 

On  the  surface  of  gelatine  a  delicate  growth  appears  along 
the  track  of  the  needle,  with  round  colonies  appearing  at  the 
edges  of  the  growth.  It  does  not  liquefy  gelatine.  It 
grows  best  at  blood-heat  ;  but  much  above  that  it  fails  to 
produce  its  fermentation,  and  it  ceases  to  grow  under  10°  C. 
It  inverts  milk-sugar  and  changes  it  to  dextrose,  from  which 
it  then  produces  lactic  acid.  Sugars  do,  however,  differ 
considerably  in  the  degrees  to  which  they  respond  to  the 
influence  of  the  lactic  ferment,  and  some  which  are  readily 
changed  by  the  alcoholic  ferment  are  untouched  by  the 
Bacillus  acidi  lactici.  It  will  be  necessary  to  refer  again  to 
this  micro-organism  when  we  come  to  speak  of  milk  and 
other  dairy  products. 

Van  Laer  has  described  a  saccharobacillus  which  produces 
lactic  acid  amongst  other  products,  and  brings  about  a 
characteristic  disease  in  beer,  named  tourne.  The  liquid 


132  BACTERIA 

gradually  loses  its  brightness  and  assumes  a  bad  odour  and 
disagreeable  taste.  The  bacillus  is  a  facultative  anaerobe. 
A  number  of  workers  have  separated  organisms,  having  a 
lactic  acid  effect,  which  diverge  considerably  from  the 
orthodox  type  of  lactic  acid  bacillus.  This  is  but  further 
evidence  of  a  fact  to  which  reference  has  been  made :  that 
nomenclature  restricted  to  one  individual  has  now  become 
adapted  to  a  family. 

4.   Butyric  Acid  Fermentation. 

Cause,  Bacillus  butyricus  and  B.  amylobacter  ;  medium,  milk,  butter,  sugar 
and  starch  solutions,  glycerine  ;  result,  butyric  acid. 

When  sugars  are  broken  down  by  the  Bacillus  acidi  lactici 
the  lactic  acid  resulting  may,  under  the  influence  of  the 
butyric  ferment,  become  converted  into  butyric  acid,  car- 
bonic acid,  and  hydrogen.  Neither  butyric  acid  nor  lactic 
acid  is  as  commonly  used  as  alcohol  or  vinegar.  Both,  like 
vinegar,  can  be  manufactured  chemically,  but  this  is  rarely 
practised.  Butyric  acid  is  a  common  ingredient  in  old  milk 
and  butter,  and  its  production  by  bacteria  is  historically 
one  of  the  first  bacterial  fermentations  understood.  More- 
over, in  its  investigation  Pasteur  first  brought  to  light  the 
fact  that  certain  organisms  acted  only  in  the  absence  of 
oxygen.  In  studying  a  drop  of  butyric  fermenting  fluid,  it 
was  observed  that  the  organisms  at  the  edge  of  the  drop 
were  motionless  and  apparently  dead,  whilst  in  the  central 
portion  of  the  drop  the  bacilli  were  executing  those  active 
movements  which  are  characteristic  of  their  vitality.  To 
Pasteur's  mind  this  at  once  suggested  what  he  was  able  later 
to  demonstrate,  namely,  that  these  bacilli  were  paralysed  by 
contact  with  oxygen.  When  he  passed  a  stream  of  air 
through  a  flask  containing  a  liquid  in  butyric  fermentation, 
he  observed  the  process  slacken  and  eventually  cease.  So 
were  discovered  the  anaerobic  micro-organisms.  The  aerobic 


BACTERIA   AND  FERMENTATION  133 

ferments  give  rise  to  oxidation  of  certain  products  of  decom- 
position ;  the  anaerobic  organisms,  on  the  other  hand,  only 
commence  to  grow  when  the  aerobic  have  used  up  all  the 
available  oxygen.  Thus  in  such  fermentations  certain  bodies 
(carbohydrates,  fatty  acids,  etc.)  undergo  decomposition,  and 
by  oxidation  become  carbonic  acid  gas,  and  the  remainder  is 
left  as  a  "  reduced  "  product  of  the  whole  process.  Hence 
sometimes  this  is  termed  fermentation  by  reduction.  The 
chemical  formula  of  this  butyric  reaction  may  be  expressed 
thus: — 

C6H12O6  (by  simple  decomposition)  =  2  C3H6O3- 
Glucose,  Lactic  acid. 

which  is  followed  by  the  fermentation  of  the  lactic  acid : — 
2  C3H603  =  C4H802  +  2  C02  +  2  H2. 

Lactic  acid.        Butyric  acid.      Carbonic  Free  hydrogen. 

acid  gas. 

Bacillus  Butyricus.  Long  and  short  rods,  generally 
straight,  with  rounded  ends,  single  or  in  chains,  reproduc- 
ing themselves  both  by  fission  and  spores,  and  sometimes 
growing  out  into  long  threads,  actively  motile,  anaerobic, 


B.  BUTYRICUS 


and  liquefying.  The  spores  are  widely  distributed  in 
nature,  and  grow  readily  on  fleshy  roots,  old  cheese,  etc. 
The  favourable  temperature  is  blood-heat,  and  on  liquid 
media  they  produce  a  pellicle.  The  resistant  spores  are 


134  BACTERIA 

irregularly  placed  in  the  rod,  and  may  cause  considerable 
variations  in  morphology.  The  culture  gives  off  a  strong 
butyric  acid  odour.  It  grows  most  readily  at  a  temperature 
of  about  40°  C. 

Although,  according  to  Pasteur's  researches,  the  butyric 
acid  ferment  performs  its  functions  anaerobically,  many 
butyric  organisms  can  act  in  the  presence  of  oxygen,  and 
yield  somewhat  different  products. 

All  of  them,  however,  ferment  most  actively  at  a  temper- 
ature at  or  about  blood-heat,  and  the  spores  are  able  to 
withstand  boiling  for  from  three  to  twenty  minutes  (Fitz). 
It  will  be  observed  that  as  in  lactic  acid  fermentation  so  in 
butyric,  the  results  are  not  due  to  one  species  only. 

5.  Ammoniacal  Fermentation  (see  under  Soil). 

Diseases  in  Beer.  We  have  seen  how  a  knowledge  of  fer- 
mentation has  been  compiled  by  a  large  number  of  workers. 
Spallanzani,  Schwann,  Pasteur,  and  Hansen  all  made  epoch- 
making  contributions.  In  the  same  way  the  investigations 
of  diseases  in  beers  and  wines  were  carried  out  by  many  ob- 
servers, and  were  closely  connected  with  those  relating  to 
spontaneous  generation  and  mixed  cultures  of  bacteria  in 
fermentation.  These  so-called  "  diseases  "  are  analogous 
to  the  taints  occurring  in  milk  and  due  to  fermentations. 
Turning  (tourne),  turbidity,  ropiness,  bitterness,  acidity, 
mouldiness,  are  all  terms  used  to  describe  these  diseases. 
They  are  chiefly  brought  about  by  four  agencies : — 

1.  Bacteria. 

2.  Mixed  yeasts. 

3.  "  Wild  "  yeasts. 

4.  Moulds. 

To  each  species  of  wild  yeast  there  belongs  some  taint- 
producing  power  in  the  fermentations  for  which  it  is  respon- 
sible. Saccharomyces  ellipsoideus  II.  and  5.  pastorianus  I. , 
///.,  are  such  yeasts;  they  only  produce  their  diseases  when 
introduced  at  the  commencement  of  the  fermentation. 


BACTERIA    AND  FERMENTATION 


135 


Saccharomyces  pastorianus  I.  is  a  low  fermentative  yeast 
in  elongated  cells,  producing  a  bitter  taste  to  beer  and  an 
unpleasant  odour.  It  can  also  produce  turbidity.  5.  pastor- 
ianus III.  produces  turbidity,  and  5.  ellipsoideus  II.  has  a 
similar  effect. 

In  1883  Hansen  demonstrated  that  the  much-dreaded 
turbidity  and  disagreeable  tastes  and  smells  in  beer  may  be 
due  to  mixture  of  two  yeasts,  each  of  which  by  itself  gives 
a  faultless  product. 

Industrial  Application  of  Bacterial  Ferments.  From  what 
has  been  said  we  trust  it  has  been  made  evident  that  bac- 
teriology has  a  place  of  ever-increasing  importance  in  regard 
to  fermentative  processes.  Not  only  have  the  caus'al  agents 
of  various  fermentations  been  isolated  and  studied,  but  from 
their  study  practical  results  follow.  The  question  of  pure 
cultures  alone  is  one  of  practical  importance ;  the  recognition 
of  the  causes  of  "  diseases  "  of  beer  is  another. 

We  cannot  enter  into  a  full  discussion  of  the  role  of  bac- 
teria in  industrial  processes,  but  several  of  the  chief  directions 
may  be  pointed  out.  Without  exception,  bacteria  have  a 
part  in  them  on  account  of  their  powers  of  fermentation.  In 
securing  their  food,  bacteria  break  down  material,  and  bring 
about  chemical  and  physical  change.  The  power  which 
organisms  have  of  chemically  destroying  compounds  is  in 
itself  of  little  importance,  but  the  products  which  arise  as  a 
result  are  of  an  importance  in  the  world  which  has  not 
hitherto  been  recognised.  We  have  used  bacteria  abund- 
antly in  the  past,  but  we  have  not  perceived  that  we  were 
doing  so.  The  maceration  industries  may  be  mentioned  as 
illustrative  of  this  use  without  acknowledgment.  The  flax 
stem  is  made  up  of  cellular  substance,  flax  fibres,  and  wood 
fibres ;  the  later  are  of  no  service  in  the  making  of  linen,  but 
the  whole  is  bound  together  by  a  gummy,  resinous  sub- 
stance. Now  this  connective  element  is  got  rid  of  in  the 
process  of  retting.  There  is  dew-retting  and  water-retting. 


136  BA  CTERIA 

The  former  is  practised  in  Russia,  and  consists  in  spreading 
the  flax  on  the  grass  and  exposing  it  to  the  influence  of  dew 
rain,  air,  and  light.  The  result  is  a  soft  and  silky  fibre. 
Water-retting  is  accomplished  by  means  of  steeping  the  flax 
in  bundles,  roots  downwards,  in  tanks  or  ponds.  In  ten  to 
fourteen  days,  according  to  the  weather,  fermentation  sets 
in,  and  breaks  the  "  shore  "  or ."  shive  "  from  the  fibre,  and 
the  process  is  complete.  This  is  always  done  by  the  aid 
of  bacteria,  which,  under  the  favourable  circumstances,  mul- 
tiply rapidly,  and  cause  decomposition  of  the  pectin  resinous 
matter.  The  same  operation  occurs  in  jute  and  hemp. 
Sponges,  too,  are  cleared  in  this  manner  by  the  rotting  of 
the  organic  matter  in  their  interstices.  The  preparation  of 
indigo  from  the  indigo  plant  is  brought  about  by  a  special 
bacterium  found  on  the  leaves.  If  the  leaves  are  sterilised, 
no  fermentation  occurs,  and  no  indigo  is  formed.  Tobacco- 
curing  is  also  in  part  due  to  decomposition  bacteria,  and 
several  bacteriologists  have  experimented  independently  in 
fermenting  tobacco  leaves  by  the  action  of  pure  cultures 
obtained  from  tobacco  of  the  finest  quality. 

In  all  these  applications  we  have  advanced  only  the  first 
stage  of  the  journey.  Nevertheless,  here,  as  in  nature  on  a 
big  scale  in  the  formation  of  fertile  soils  and  coal-measures, 
we  find  bacteria  silently  at  work,  achieving  great  ends  by 
co-operating  in  countless  hordes. 


CHAPTER  V 
BACTERIA  IN   THE  SOIL 

OURFACE  soils  and  those  rich  in  organic  matter  supply 
O  a  varied  field  for  the  bacteriologist.  Indeed,  it  may 
be  said  that  the  introduction  of  the  plate  method  of  culture 
and  the  improved  facilities  for  growing  anaerobic  micro- 
organisms have  opened  up  possibilities  of  research  into  soil 
microbiology  unknown  to  previous  generations  of  workers. 
From  the  nature  of  bacteria  it  will  be  readily  understood 
that  their  presence  is  affected  by  geological  and  physical 
conditions  of  the  soil,  and  in  all  soils  only  within  a  few  feet 
of  the  surface.  As  we  go  down  below  two  feet,  bacteria 
become  less,  and  below  a  depth  of  five  or  six  feet  we  find 
only  a  few  anaerobes.  At  a  depth  of  ten  feet,  and  in  the 
' '  ground  water  region, ' '  bacteria  are  scarce  or  absent.  This 
is  held  to  be  due  to  the  porosity  of  the  soil  acting  as  a  filter- 
ing medium.  Regarding  the  numbers  of  micro-organisms 
present  in  soil,  no  very  accurate  standard  can  be  obtained. 
Ordinary  earth  may  yield  anything  from  10,000  to  5,000,- 
ooo  per  gram,  whilst  from  polluted  soil  even  100,000,000  per 
gram  have  been  estimated.  These  figures  are  obviously 
only  approximate,  nor  is  an  exact  standard  of  any  great 
value.  Nevertheless,  Frankel,  Beumer,  Miquel,  and  Mag- 
giora  have,  as  the  result  of  experiments,  arrived  at  a  num- 
ber of  conclusions  respecting  bacteria  in  soil  which  are  of 
much  more  practical  use.  From  these  results  it  appears 
that,  in  addition  to  the  "  ground  water  region  "  being  free, 

137 


138  BACTERIA 

or  nearly  so,  virgin  soils  contain  much  fewer  than  cultivated 
lands,  and  these  latter,  again,  fewer  than  made  soils  and 
inhabited  localities.  In  cultivated  lands  the  number  of 
organisms  augments  with  the  activity  of  cultivation  and  the 
strength  of  the  fertilisers  used.  In  all  soils  the  maximum 
occurs  in  July  and  August. 

But  the  condition  which  more  than  all  others  controls  the 
quantity  and  quality  of  the  contained  bacteria  is  the  degree 
and  quality  of  the  organic  matter  in  the  soil.  The  quantity 
of  organic  matter  present  in  soil  having  a  direct  effect  upon 
bacteria  will  be  materially  increased  by  placing  in  soil  the 
bodies  of  men  and  animals  after  death.  Dr.  Buchanan 
Young  two  or  three  years  ago  performed  some  experiments 
to  discover  to  what  degree  the  soil  bacteria  were  affected  by 
these  means.  '  The  number  of  micro-organisms  present  in 
soil  which  has  been  used  for  burial  purposes,"  he  concludes, 
"  exceeds  that  present  in  undisturbed  soil  at  similar  level, 
and  this  excess,  though  apparent  at  all  depths,  is  most 
marked  in  the  lower  reaches  of  the  soil."  l  The  numbers 
were  as  follows: — 

Virgin  soil,  4  ft.  6  in.  =  53,436  m.o.  per  gram  of  soil. 
Burial  soil  (8  years),  4  ft.  6  in.=  363,411  m.o.  per  gram  of  soil. 
(3     "     ),  6ft.  6  in.=  722,751 

Methods  of  Examination  of  Soil.  Two  simple  methods  are 
generally  adopted.  The  first  is  to  obtain  a  qualitative  esti- 
mation of  the  organisms  contained  in  the  soil.  It  consists 
simply  in  adding  to  test-tubes  of  liquefied  gelatine  or  broth 
a  small  quantity  of  the  sample,  finely  broken  up  with  a 
sterile  rod.  The  test-tubes  are  now  incubated  at  37°  C.  and 
22°  C.,  and  the  growth  of  the  contained  bacteria  observed 
in  the  test-tube,  or  after  a  plate  culture  has  been  made. 
The  second  plan  is  adopted  in  order  to  secure  more  accurate 
quantitative  results.  One  gram  or  half-gram  of  the  sample 

1  Proc.  Royal  Soc.  of  Edin.,  xxxvii.,  pt.  iv.,  p.  759. 


BACTERIA   IN   THE   SOIL  139 

is  weighed  on  the  balance,  and  then  added  to  1000  cc.  of 
distilled  sterilised  water  in  a  sterilised  flask,  in  which  it  is 
thoroughly  mixed  and  washed.  From  either  of  these  two 
different  sources  it  is  now  possible  to  make  sub-cultures  and 
plate  cultures.  The  procedure  is,  of  course,  that  described 
under  the  examination  of  water  (p.  41  et  seq.\  and  Petri's 
dishes,  Koch's  plates,  or  Esmarch's  roll  cultures  are  used. 
Many  of  the  commoner  bacteria  in  soil  will  thus  be  detected 
and  cultivated.  But  it  is  obvious  that  this  by  no  means 
covers  the  required  ground.  It  will  be  necessary  for  us  here 
to  consider  the  methods  generally  adopted  for  growing  an- 
aerobic bacteria,  that  is  to  say  those  species  which  will  not 
grow  in  the  presence  of  oxygen.  This  anaerobic  difficulty 
may  be  overcome  in  a  variety  of  ways. 

1.  The  air  contained  in  the  culture  tube  may  be  removed 
by  ebullition  and  rapid  cooling.     And  whilst  this  may  ac- 
curately produce  a  vacuum,  it  is  far  from  easy  to  introduce 
the  virus  without  also  reintroducing  oxygen. 

2.  The  oxygen  may  be  displaced  by  some  other  gas,  and 
though  coal-gas,  nitrogen,  and  carbon  dioxide  may  all  be 
.used  for  this  purpose,  it  has  become  the  almost  universal 
practice  to  grow  anaerobes  in  hydrogen.     The  production  of 
the  hydrogen  is  readily  obtained  by  Kipp's  or  some  other 
suitable  apparatus  for  the  generation  of  hydrogen  from  zinc 
and  sulphuric  acid.     The  free  gas  is  passed  through  various 
wash-bottles   to   purify  it  of   any   contaminations.      Lead 
acetate  (i-io  per  cent,  water)  removes  any  traces  of  sul- 
phuretted hydrogen,  silver  nitrate  (i-io)  doing  the  same  for 
arseniated  hydrogen ;  whilst  a  flask  of  pyrogallic  acid  will 
remove  any  oxygen.     It  is  not  always  necessary  to  have 
these  three  purifiers  if  the  zinc  used  in  the  Kipp's  apparatus  is 
pure.     Occasionally  a  fourth  flask  is  added  of  distilled  water, 
and  this  or  a  dry  cotton  wool  pledget  in  the  exit  tube  will 
ensure  germ-free  gas.     From  the  further  end  of  the  exit 
tube  of  the  Kipp's  apparatus  an  india-rubber  tube  will  carry 


140 


BACTERIA 


the  hydrogen  to  its  desired  destination.  With  some  it  is  the 
custom  to  place  anaerobic  cultures  in  test-tubes,  and  the 
test-tubes  in  a  large  flask  having  a  two-way  tube  for  en- 
trance and  exit  of  the  hydrogen ;  others  prefer  to  pass  the 
hydrogen  immediately  into  a  large  test-tube  containing 

the  culture  (Frankel's  method). 
Either  method  ends  practically 
the  same,  and  the  growth  of 
the  culture  in  hydrogen  is  read- 
ily observed.  Yet  another  plan 
is  to  use  a  yeast  flask,  and  after 
having  passed  the  hydrogen 
through  for  about  half  an  hour, 
the  lateral  exit  tube  is  dipped 
into  a  small  flask  containing 
mercury.  The  entrance  tube  is 
now  sealed,  and  the  whole  ap- 


Cultfvation  Flask 


The  Zinc  and  Acid  Vessel 


KIPP'S  APPARATUS 

For  the  Production  of  Hydrogen 


paratus  placed  in  the  incubator.     The  interior  containing 
the  culture  is  filled  with  an  atmosphere  of  hydrogen.     No 


BACTERIA    IN   THE   SOIL 


141 


u 


oxygen  can  obtain  entrance  through  the  sealed  entrance 
tube,  or  through  the  exit  tube  immersed  in  mercury.  Yet 
through  this  latter  channel  any  gases  produced  by  the  cult- 
ure could  escape  if  able  to  produce  sufficient  pressure. 

3.  The  Absorption  Method.     Instead  of  adding  hydrogen 
to  the  tube  or  flask  containing  the  anaerobic  culture,  it  is 

feasible  to  add  to  the  medium 
some  substances,  like  glucose 
or  pyrogallic  acid,  which  will 
absorb  the  oxygen  which  is 
present,  and  thus  enable  the 
anaerobic  requirement  to  be 
fulfilled.  To  various  media — 
gelatine,  agar,  or  broth  (the 
latter  used  for  obtaining  the 
toxins  of  anaerobes)  —  2  per 
cent,  of  glucose  may  be  added. 
Pyrogallic  acid,  or  pyrogallic 
acid  one  part  and  20  per  cent, 
caustic  potash  one  part,  is  also 
readily  used  for  absorptive  pur- 
poses. A  large  glass  tube  of 
25  cc.  height,  named  a  Buch- 
ner's  cylinder,  having  a  con- 
striction near  the  bottom,  is  taken;  and  about 
two  drachms  of  the  pyrogallic  solution  are  placed 
in  the  bottom  of  it.  A  test-tube  containing  the 
culture  is  now  lodged  in  the  upper  part  above  the 
constriction.  The  apparatus  is  now  placed  in 
the  incubator  at  the  desired  temperature,  and  the 
contained  culture  grows  under  anaerobic  condi- 
tions. As  the  pyrogallic  solution  absorbs  the 
oxygen  it  assumes  a  darker  tint. 

4.  Mechanical  Methods.     These  include  various 
ingenious  tricks  for  preventing  an  admittance  of  oxygen  to 


FRANKEL'S  TUBE 

For  Cultivation  of 
Anaerobes 


BUCHNER'S 
TUBE 

For  Cultivation 
of  Anaerobes 


142  BACTERIA 

the  culture.  An  old-fashioned  one  was  to  plate  out  the 
culture  and  protect  it  from  the  air  by  covering  it  with  a 
plate  of  mica.  A  more  serviceable  mode  is  to  inoculate, 
say,  a  tube  of  agar  with  the  anaerobic  organism,  and  then 
pour  over  the  culture  a  small  quantity  of  melted  agar,  which 
will  readily  set,  and  so  protect  the  culture  itself  from  the 
air.  Oil  may  be  used  instead  of  melted  agar.  Another 
mechanical  method  is  to  make  a  deep  inoculation  and  then 
melt  the  top  of  the  medium  over  a  bunsen  burner,  and  thus 
close  the  entrance  puncture  and  seal  it  from  the  air. 

5.  Absorption  of  Oxygen   by   an   Aerobic    Culture.     This 
method  takes  advantage  of  the  power  of  absorption  of  cer- 
tain aerobic  bacteria,  which  are  planted  over  the  culture  of 
the  anaerobic  species.     It   is  not   practically  satisfactory, 
though  occasionally  good  results  have  been  obtained. 

6.  Lastly,  there  is  the  Air-pump  Method.     By  this  means 
it  is  obviously  intended  to  extract  air  from  the  culture  and 
seal  of  it  in  vacua.     The  culture  tubes  are  connected  with 
the  air-pump,  and  exhausted  as  much  as  possible. 

Of  these  various  methods  it  is  on  the  whole  best  to  choose 
either  the  hydrogen  method,  the  vacuum,  or  the  plan  of 
absorption  by  grape-sugar  or  pyrogallic.  In  anaerobic  plate 
cultures  grape-sugar  agar  plus  0.5  per  cent,  of  formate  of 
soda  may  be  used.  The  poured  inoculated  plate  should  be 
placed  over  pyrogallic  solution  under  a  sealed  bell-glass  and 
incubated  at  37°  C.  Pasteur,  Roux,  Joubert,  Chamberland, 
Esmarch/Kitasato,  and  others  have  introduced  special  ap- 
paratus to  facilitate  anaerobic  cultivation,  but  the  principles 
adopted  are  those  which  have  been  mentioned. 

THE  QUALITATIVE  ESTIMATION  OF  BACTERIA  IN  THE  SOIL 

We  may  now  turn  to  consider  the  species  of  bacteria 
found  in  the  interstices  of  soil.  They  may  be  classified  in 
five  main  groups.  The  division  is  somewhat  artificial,  but 
convenient : 


BACTERIA   IN   THE   SOIL 


143 


I.  The  Denitrifying  Bacteria.  A  group  whose  function 
has  been  elucidated  in  recent  years  (largely  by  the  investiga- 
tions of  Professor  Warington)  are  held  responsible  for  the 
breaking  down  of  nitrates.  With  these  may  be  associated 


A  METHOD  OF  GROWING  CULTIVATIONS  IN  A  VACUUM 
OVER  PYROGALLIC  SOLUTION 

the  Decomposition  or  Putrefactive  Bacteria,  which  break 
down  complex  organic  products  other  than  nitrates  into 
simpler  bodies. 

2.    The  Organisms  of  Nitrification.     To  this  group  belong 


144  BACTERIA 

the  two  chief  types  of  nitrifying  bacteria,  viz.,  those  which 
oxidise  ammonia  into  nitrites,  and  those  which  change 
nitrites  into  nitrates. 

3.  The  Nitrogen-fixing  Bacteria,    found    mainly    in    the 
nodules  on  the  rootlets  of  certain  plants. 

4.  The  Common  Saprophytic  Bacteria,  whose  function  is  at 
present    but    imperfectly    known.      Many   are   putrefactive 
germs. 

5.  The  Pathogenic  Bacteria.     This  division  includes  the 
three  types,  tetanus,   malignant  oedema,   and  quarter  evil. 
Under  this  heading  we  shall  also  have  to  consider  in  some 
detail  the  intimate  relation  between  the  soil  and  such  im- 
portant bacterial  diseases  as  tubercle  and  typhoid. 

To  enable  us  to  appreciate  the  work  which  the  "  economic 
bacteria"  perform,  it  will  be  necessary  to  consider  shortly 
the  place  they  occupy  in  the  economy  of  nature.  This  may 
be  perhaps  most  readily  accomplished  by  studying  the 
accompanying  table  (p.  145). 

The  threefold  function  of  plant  life  is  nutrition,  assimila- 
tion, and  reproduction  :  the  food  of  plants,  the  digestive  and 
storage  power  of  plants,  and  the  various  means  they  adopt 
for  multiplying  and  increasing  their  species.  With  the  two 
latter  we  have  little  concern  in  this  place.  Respecting  the 
nutrition  of  plant  life,  it  is  obvious  that,  like  animals,  they 
must  feed  and  breathe  to  maintain  life.  Plant  food  is  of 
three  kinds,  viz.,  water,  chemical  substances,  and  gas.  Water 
is  an  actual  necessity  to  the  plant  not  only  as  a  direct  food 
and  food-solvent,  but  as  the  vehicle  of  important  inorganic 
materials.  The  hydrogen,  too,  of  the  organic  compounds 
is  obtained  from  the  decomposition  of  the  water  which  per- 
meates every  part  of  the  plant,  and  is  derived  by  it  from  the 
soil  and  from  the  aqueous  vapour  in  the  atmosphere.  The 
chief  chemical  substances  of  which  vegetable  protoplasm 
is  constituted  are  six,  viz,  potassium,  magnesium,  cal- 
cium, iron,  phosphorous,  and  sulphur.  These 


BACTERIA   IN   THE   SOIL 


145 


A    SCHEME    SHOWING   THE    PLACE   AND   FUNCTION   OF   THE 
ECONOMIC    MICRO-ORGANISMS   FOUND    IN   SOIL 

Water  Chemical  Substances  Gases 

[Nitrates,  etc.]  [CO2,H,N,O] 

\  / 


PLANT  LIFE 

I 

T 


Carbohydrates         Fats  Proteids  Vegetable        Mineral        Water 

[albumoses,  sugar,  [bodies  containing        Acids  Salts 

starch,  etc.]  Nitrogen] 


ANIMAL  LIFE 

Gases  [CO2,  etc.]       Water  Urea,  Albuminoids,  Nitrogen  in  many 

Ammonia  compounds,  etc.        forms  locked  up 

in  the  body 


PUTREFACTIVE    AND    DENITRIFYING    BACTERIA 


r          ~T~ 

Free  Nitrogen        Gases  [CO2] 


NITROGEN-FIXING 
BACTERIA 

[In^oil  and  in  the  nodules 
le  rootlets  of  Legu- 


Water          Ammonia          [Nitrites] 
and  other  elements 
of  broken-down 
complex  bodies. 

NITRIFYING    BACTERIA 
I 

Nitrites[= Nitrous  organism 

|  (Nitrosomans)] 

Nitrates[  =  Nitric  organism 
(Nitrosomonas)J 
[In    soil    and 
available  for 
plant  life] 


146  BACTERIA 

elements  do  not  enter  the  plant  as  such,  but  combined  with 
other  substances  or  dissolved  in  water.  Potassium  occurs  in 
salt  form  combined  with  various  organic  acids  (tartaric, 
oxalic,  etc.),  calcium  and  magnesium  as  salts  of  lime  and 
magnesia  in  combination  both  with  organic  and  inorganic 
acids.  Iron  contributes  largely  to  the  formation  of  the 
green  colouring  matter  of  plants,  and  is  also  derived  from 
the  soil.  Phosphorus,  one  of  the  chief  constituents  of  seeds, 
generally  occurs  as  phosphate  of  lime.  Sulphur,  which  is 
an  important  constituent  of  albumen,  is  derived  from  the 
sulphates  of  the  soil.  In  addition  to  the  above,  there  are 
other  elements,  sometimes  described  as  non-essential  con- 
stituents of  plants.  Amongst  these  are  silica  (to  give  stiff- 
ness), sodium,  chlorine,  iodine,  bromine,  etc.  All  these 
elements  contribute  to  the  formation  or  quality  of  the 
protoplasm  of  plants. 

The  gases  essential  to  plants  are  four:  Carbon  dioxide 
(carbonic  acid),  Hydrogen,  Oxygen,  and  Nitrogen.  By  the 
aid  of  the  green  chlorophyll  corpuscles,  and  under  the  influ- 
ence of  sunlight,  we  know  that  leaves  absorb  the  carbon 
dioxide  of  the  atmosphere,  and  effect  certain  changes  in  it. 
The  hydrogen,  as  we  have  seen,  is  obtained  from  the  water. 
Oxygen  is  absorbed  through  the  root  from  the  interstices 
of  the  soil.  Each  of  these  contributes  vitally  to  the  exist- 
ence of  the  plant.  The  fourth  gas,  nitrogen,  which  consti- 
tutes more  than  two  thirds  of  the  air  we  breathe  (79  per 
cent,  of  the  total  volume  and  77  per  cent,  of  the  total  weight 
of  the  atmosphere),  is,  perhaps,  the  most  important  food 
required  by  plants.  Yet,  although  this  is  so,  the  plant  can- 
not absorb  or  obtain  its  nitrogen  in  the  same  manner  in 
which  it  acquires  its  carbon — viz.,  by  absorption  through 
the  leaves — nor  can  the  plant  take  nitrogen  into  its  own  sub- 
stance by  any  means  as  nitrogen,  with  the  exception  .ef-.the 
flesh-feeding  plants  (insectivorus).  Hence,  although,  this 
gas  is  present  in  the  atmosphere  surrounding  the  plant,  the 


BACTERIA   IN   THE   SOIL  147 

plant  will  perish  if  nitrogen  does  not  exist  in  some  combined 
form  in  the  soil.  Nitrates  and  compounds  of  ammonia  are 
widely  distributed  in  nature,  and  it  is  from  these  bodies 
that  the  plant  obtains,  by  means  of  its  roots,  the  necessary 
nitrogen. 

Until  comparatively  recently  it  was  held  that  plant  life 
could  not  be  maintained  in  a  soil  devoid  of  nitrogen  or  com- 
pounds thereof.  But  it  has  been  found  that  certain  classes 
of  plants  (the  Leguminosce,  for  example),  when  they  are 
grown  in  a  soil  which  is  practically  free  from  nitrogen  at  the 
commencement,  do  take  up  this  gas  into  their  tissues.  One 
explanation  of  this  fact  is  that  free  nitrogen  becomes  con- 
verted into  nitrogen  compounds  in  the  soil  through  the 
influence  of  micro-organisms  present  there.  Another  ex- 
planation attributes  this  fixation  of  free  nitrogen  to  micro- 
organisms existing  in  the  rootlets  of  the  plant.  These  two 
classes  of  organisms,  known  as  the  nitrogen-fixing  organisms, 
will  require  our  consideration  at  a  later  stage.  Here  we 
merely  desire  to  make  it  clear  that  the  main  supply  of  this 
gas,  absolutely  necessary  to  the  existence  of  vegetable  life 
upon  the  earth,  is  drawn  not  from  the  nitrogen  of  the  at- 
mosphere, but  from  that  contained  in  nitrogen  compounds 
in  the  soil.  The  most  important  of  these  are  the  nitrates. 
Here  then  we  have  the  necessary  food  of  plants  expressed 
in  a  sentence :  water,  gases,  salts,  the  most  important  and 
essential  gas  and  some  of  the  salts  being  combined  in  nitrates. 

Plant  life  seizes  upon  its  required  constituents,  and  by 
means  of  the  energy  furnished  by  the  sun's  rays  builds  these 
materials  up  into  its  own  complex  forms.  Its  many  and 
varied  forms  fulfil  a  place  in  beautifying  the  world.  But 
their  contribution  to  the  economy  of  nature  is,  by  means  of 
their  products,  to  supply  food  for  animal  life.  The  products 
of  plant  life  are  chiefly  sugar,  starch,  fat,  and  proteids. 
Animal  life  is  not  capable  of  extracting  its  nutriment  from 
soil,  but  it  must  take  the  more  complex  foods  which  have 


148  BACTERIA 

already  been  built  up  by  vegetable  life.  Again,  the  com- 
plementary functions  of  animal  and  vegetable  life  are  seen 
in  the  absorption  by  plants  of  one  of  the  waste  materials  of 
animals,  viz.,  carbonic  acid  gas.  Plants  abstract  from  this 
gas  carbon  for  their  own  use,  and  return  the  oxygen  to  the 
air,  which  in  its  turn  is  of  service  to  animal  life. 

By  animal  activity  some  of  these  foods  supplied  by  the 
vegetable  kingdom  are  at  once  decomposed  into  carbonic 
acid  gas  and  water,  which  goes  back  to  nature.  Much, 
however,  is  built  up  still  further  into  higher  and  higher  com- 
pounds. The  proteids  are  converted  by  digestion  into 
alburnoses  and  peptones,  ultimately  entirely  into  peptones; 
these  in  their  turn  are  reconverted  into  proteids,  and  be- 
come assimilated  as  part  of  the  living  organism.  In  time 
they  become  further  changed  into  carbonic  acid,  sulphuric 
acid,  water,  and  certain  not  fully  oxidised  products,1  which 
contain  the  nitrogen  of  the  original  proteid.  In  the  table 
these  bodies  have  been  represented  by  one  of  their  chief 
members,  viz.,  urea. 

It  is  clear  that  there  is  in  all  animal  life  a  double  process 
continually  going  on ;  there  is  a  building  up  (anabolism,  as- 
similation), and  there  is  a  breaking  down  (katabolism,  dis- 
similation). These  processes  will  not  balance  each  other 
throughout  the  whole  period  of  animal  life.  We  have,  as 
possibilities,  elaboration,  balance,  degeneration ;  and  the 
products  of  animal  life  will  differ  in  degree  and  in  substance 
according  to  which  period  is  in  the  predominance.  These 
products  we  may  subdivide  simply  into  excretions  during 
life  and  final  materials  of  dissolution  after  death,  both  of 
which  may  be  used  more  or  less  immediately  by  other  forms 
of  animal  or  vegetable  life,  or  mediately  after  having  passed 
to  the  soil.  We  may  shortly  summarise  the  final  products 
of  animal  life  as  carbonic  acid,  water,  and  nitrogenous  rem- 
nants. These  latter  will  occur  as  urea,  new  albumens,  com- 

1  E.  A.  Schafer,  Text-book  of  Physiology,  vol.  i.,  p.  25  (W.  D.  Halliburton). 


BACTERIA   IN   THE   SOIL  149 

pounds  of  ammonia,  and  nitrogen  compounds  of  great  com- 
plexity stored  up  in  the  tissues  and  body  of  the  animal. 
The  carbonic  acid,  water,  and  other  simple  substances  like 
them  will  return  to  nature  and  be  of  immediate  use  to  vege- 
table life.  But  otherwise  the  cycle  cannot  be  completed, 
for  the  more  complex  bodies  are  of  no  service  as  such  to 
plants  or  animals. 

i.  In  order  that  this  complex  material  should  be  of  service 
in  the  economy  of  nature,  and  its  constituents  not  lost,  it  is 
necessary  that  it  should  be  broken  down  again  into  simpler 
conditions.  This  prodigious  task  is  accomplished  by  the 
agency  of  two  groups  of  organisms,  the  decomposition  and 
denitrifying1  bacteria.  The  organisms  associated  with  de- 
composition processes  are  numerous;  some  denitrify  as  well 
as  break  down  organic  compounds.  This  group  will  be 
referred  to  under  "  Saprophytic  Bacteria."  The  reduction 
by  the  denitrifying  bacteria  may  be  simply  from  nitrate  to 
nitrite,  or  from  nitrate  to  nitric  or  nitrous  oxide  gas,  or  in- 
deed to  nitrogen  itself.  In  all  these  processes  of  reduction 
the  rule  is  that  a  loss  of  nitrogen  is  involved.  How  that 
free  nitrogen  is  brought  back  again  and  made  subservient  to 
plants  and  animals  we  shall  understand  at  a  later  stage. 

Professor  Warington  has  again  recently  set  forth  the  chief 
facts  known  of  this  decomposition  process.3  That  the 
action  in  question  only  occurs  in  the  presence  of  living 
organisms  was  first  established  by  Mensel  in  1875  in  natural 
waters,  and  by  Macquenne  in  1882  in  soils.  If  all  living 
organisms  are  destroyed  by  sterilisation  of  the  soil,  denitri- 
fication  cannot  take  place,  nor  can  vegetable  life  exist. 
"  Bacteria  reduce  nitrates,"  says  Professor  Warington,  "  by 
bringing  about  the  combustion  of  organic  matter  by  the 
oxygen  of  the  nitrate,  the  temperature  distinctly  rising 

1  ' '  Denitrifying  "  means  reducing  nitrates. 

2R.  Warington,  M.A.,  F.R.S.,  Journ.  Roy.  Agricultural  Soc.  Eng.,  series 
iii.,  vol.  viii.,  pt.  iv.,  pp.  577  et  seq. 


I5O  BACTERIA 

during  the  operation/'  The  reduction  to  a  nitrite  is  a  com- 
mon property  of  bacteria.  But  only  a  few  species  have  the 
power  of  reducing  a  nitrate  to  gas.  These  few  species  are, 
however,  widely  distributed.  In  1886  Gayon  and  Dupetit 
first  isolated  the  bacteria  capable  of  reducing  nitrates  to  the 
simplest  element,  nitrogen.  They  obtained  their  species 
from  sewage,  but  ten  years  later  denitrifying  bacteria  were 
isolated  from  manure.  That  soil  contains  a  number  of  these 
reducing  organisms  is  known  by  introducing  a  particle  of 
surface  soil  into  some  broth,  to  which  has  been  added  one 
per  cent,  of  nitre.  During  incubation  of  such  a  tube  gas  is 
produced,  and  the  nitrate  entirely  disappears. 

Whenever  decomposition  occurs  in  organic  substances 
there  is  a  reduction  of  compound  bodies,  and  in  such  cases 
the  putrefying  substances  obtain  their  decomposing  and 
denitrifying  bacteria  from  the  air.  The  chief  conditions 
requisite  for  bringing  about  a  loss  of  nitrogen  by  denitrifi- 
cation  are  enumerated  by  Professor  Warington  as  follows: 
(i)  the  specific  micro-organism  ;  (2)  the  presence  of  a  nitrate 
and  suitable  organic  matter;  (3)  such  a  condition  as  to 
aeration  that  the  supply  of  atmospheric  oxygen  shall  not 
be  in  excess  relatively  to  the  supply  of  organic  matter ;  (4) 
the  usual  essential  conditions  of  bacterial  growth.  "  Of 
these,"  he  says,  "  the  supply  of  organic  matter  is  by  far  the 
most  important  in  determining  the  extent  to  which  denitri- 
fication  will  take  place."  The  necessarily  somewhat  un- 
stable condition  facilitates  its  being  split  up  by  means  of 
bacteria.  The  bacteria  in  their  turn  are  ready  to  seize  upon 
any  products  of  animal  life  which  will  serve  as  their  food. 
Thus,  by  reducing  complex  bodies  to  simple  ones,  these 
denitrifying  organisms  act  as  the  necessary  link  to  connect 
again  the  excretions  of  the  animal  body,  or  after  death  the 
animal  body  itself,  with  the  soil. 

In  a  book  of  this  nature  it  has  been  deemed  advisable  not 
to  enter  into  minute  description  of  all  the  species  of  bacteria 


BACTERIA   IN   THE   SOIL  151 

mentioned.  Some  of  the  chief  are  described  more  or  less 
fully.  We  cannot,  however,  do  more  than  name  several  of 
the  chief  organisms  concerned  in  reducing  and  breaking 
down  compounds.  As  we  shall  find  in  the  bacteria  of 
nitrification,  so  also  here,  the  entire  process  is  rarely,  if  ever, 
performed  by  one  species.  There  is  indeed  a  remarkable 
division  of  labour,  not  only  between  decomposition  bacteria 
and  denitrification  bacteria,  but  between  different  species  of 
the  same  group.  Bacillus  fluorescens  non-liquefaciens,  My- 
coderma  urea,  and  some  of  the  staphylococci  break  down 
nitrates  (denitrification),  and  also  decompose  other  com- 


*:••:  .*>••: 


t 

MlCROCOCCUS    FROM    SOIL 

pound  bodies.  Amongst  the  group  of  putrefactive  bacteria 
found  in  soil  may  be  named  B.  coli,  B.  mycoides,  B.  mesen- 
tericus,  B.  liquidus,  B.  prodigiosus,  B.  ramosus,  B.  vermicu- 
lar is,  B.  liquefaciens,  and  many  members  in  the  great  family 
of  Proteus.  Some  perform  their  function  in  soil,  others  in 
water,  and  others,  again,  in  dead  animal  bodies.  Dr. 
Buchanan  Young,  to  whose  researches  in  soil  we  have  re- 
ferred, has  pointed  out  that  in  the  upper  reaches  of  burial 
soil,  where  these  bacteria  are  most  largely  present,  there  is 
as  a  result  no  excess  of  organic  carbon  and  nitrogen.  Even 
in  the  lower  layers  of  such  soil  it  is  rapidly  broken  down. 

It  will  be  observed,  from  a  glance  at  the  table,  that  the 
chief  results  of  decomposition  and  denitrification  are  as  fol- 


152  BACTERIA 

lows:  free  nitrogen,  carbonic  acid,  gas  and  water,  ammonia 
bodies,  and  sometimes  nitrites.  The  nitrogen  passes  into 
the  atmosphere,  and  is  "  lost  "  ;  the  carbonic  acid  and  water 
return  to  nature  and  are  at  once  used  by  vegetation.  The 
ammonia  and  nitrites  await  further  changes.  These  further 
changes  become  necessary  on  account  of  the  fact,  already 
discussed,  that  plants  require  their  nitrogen  to  be  in  the 
form  of  nitrates  in  order  to  use  it.  Nitrates  obviously  con- 
tain a  considerable  amount  of  oxygen,  but  ammonia  contains 
no  oxygen,  and  nitrites  very  much  less  than  nitrates.  Hence 
a  process  of  oxidation  is  required  to  change  the  ammonia 
into  nitrites  and  the  nitrites  into  nitrates. 

2.  This  oxidation  is  performed  by  the  nitrifying  micro- 
organisms, and  the  process  is  known  as  nitrification.  It 
should  be  clearly  understood  that  the  process  of  nitrifaction 
may,  so  to  speak,  dovetail  with  the  process  of  denitrification. 
No  exact  dividing  line  can  be  drawn  between  the  two,  al- 
though they  are  definite  and  different  processes.  In  a 
carcass,  for  example,  both  processes  may  be  going  on  con- 
comitantly;  so  also  in  manure.  There  is  no  hard  and  fast 
line  to  be  drawn  in  the  present  state  of  our  knowledge. 
Other  organisms  beside  the  true  nitrification  bacteria  may 
be  playing  a  part,  and  it  is  impossible  exactly  to  measure 
the  action  of  the  latter,  where  they  began  and  where  the 
preliminary  attack  upon  the  nitrogenous  compounds  term- 
inated. In  all  cases,  however,  according  to  Professor  War- 
ington,  the  formation  of  ammonia  has  been  found  to  precede 
the  formation  of  nitrous  or  nitric  acid. 

It  was  Pasteur  who  (in  1862)  first  suggested  that  the  pro- 
duction of  nitric  acid  in  soil  might  be  due  to  the  agency  of 
germs,  and  it  is  to  Schlosing  and  Miintz  that  the  credit  be- 
longs for  first  demonstrating  (in  1877)  that  the  true  nature 
of  nitrification  depended  upon  the  activity  of  a  living  micro- 
organism. Partly  by  Schlosing  and  Miintz  and  partly  by 
Warington  (who  was  then  engaged  in  similar  work  at  Roth- 


BACTERIA   IN   THE   SOIL  153 

amsted),  it  was  later  established  (i)  that  the  power  of  nitri- 
fication could  be  communicated  to  substances  which  did  not 
hitherto  nitrify  by  simply  seeding  them  with  a  nitrified  sub- 
stance, and  (2)  that  the  process  of  nitrification  in  garden 
soil  was  entirely  suspended  by  the  vapour  of  chloroform  or 
carbon  disulphide.  The  conditions  for  nitrification,  the 
limit  of  temperature,  and  the  necessity  of  plant  food,  have 
furnished  additional  proof  that  the  process  is  due  to  a  living 
organism.  These  conditions  are  briefly  as  follows : 

1.  Food  (of  which  phosphates  are  essential  constituents). 
"  The  nitrifying  organism  can  apparently  feed  upon  organic 
matter,  but  it  can  also,  apparently  with  equal  ease,  develop 
and  exercise  all  its  functions  with  purely  inorganic  food  " 
(Warington). 

Winogradsky  prepared  vessels  and  solutions  carefully  puri- 
fied from  organic  matter,  and  these  solutions  he  sowed  with 
the  nitrifying  organism,  and  found  that  they  flourished. 
Professor  Warington  has  employed  the  acid  carbonates  of 
sodium  and  calcium  with  distinct  success  as  ingredients  of  an 
ammoniacal  solution  undergoing  nitrification. 

2.  The  next  condition  of  nitrification  is  the  presence  of 
oxygen.     Without  it  the  reverse  process,   denitrification, 
occurs,  and  instead  of  a  building  up  we  get  a  breaking 
down,  with  an  evolution  of  nitrogen  gas.     The  amount  of 
oxygen  present  has  an  intimate  proportion  to  the  amount 
of  nitrification,  and  with  16  to  21  per  cent,  of  oxygen  present 
the  nitrates  are  more  than  four  times  as  much  as  when  the 
smallest  quantity  of  oxygen  is  supplied.     The  use  of  tillage 
in  promoting  nitrification  is  doubtless  in  part  due  to  the 
aeration  of  the  soil  thus  obtained. 

3.  A  third  condition  is  the  presence  of  a  base  with  which 
nitric  acid  when  formed  may  combine.     Nitrification  can 
take  place  only  in  a  feebly  alkaline  medium,  but  an  excess 
of  alkilinity  will  retard  the  process. 

4.  The  last  essential  requirement  is  a  favourable  temper- 


154  BACTERIA 

ature.  The  nitrifying  organism  can  act  at  a  temperature  as 
low  as  37°  or  39°  F.  (3-4°  C.),  but  at  a  higher  temperature 
it  becomes  much  more  active.  According  to  Schlosing  and 
Miintz,  at  54°  F.  (12°  C.)  nitrification  becomes  really  active, 
and  it  increases  as  the  temperature  rises  to  99°  F.  (37°  C.), 
after  which  it  falls.  A  high  temperature  or  a  strong  light 
are  prejudicial  to  the  process. 

We  are  now  in  a  position  to  consider  shortly  some  of  the 
characters  of  these  nitrification  bacteria.  They  may  readily 
be  divided  into  two  chief  groups,  not  in  consideration  of 
their  form  or  biological  characteristics,  but  on  account  of  the 
duties  which  they  perform.  Just  as  we  observed  that  there 
were  few  denitrifying  organisms  which  could  break  down 
ammonia  compounds  to  nitrogen  gas,  so  is  it  also  true  that 
there  are  few  nitrifying  bacteria  which  can  build  up  from 
ammonia  to  the  nitrates.  Nature  has  provided  that  this 
shall  be  accomplished  in  two  stages,  viz.,  a  first  stage  from 
ammonia  bodies  to  nitrites,  and  a  second  stage  from  nitrites 
to  nitrates.  The  agent  of  the  former  is  termed  the  nitrous 
organism,  the  latter  the  nitric  organism.  Both  are  con- 
tributing to  the  final  production  of  nitrates  which  can  be 
used  by  plant  life.' 

The  Nitrous  Organism  (Nitrosomonas).  Prior  to  Koch's 
gelatine  method  the  isolation  of  this  bacterium  proved  an 
exceedingly  difficult  task.  But  even  the  adoption  of  this 
isolating  method  seemed  to  give  no  better  results,  and  for 
an  excellent  reason :  the  nitrifying  organisms  will  not  grow 
on  gelatine.  To  Winogradsky  and  Percy  Frankland  belongs 
the  credit  of  separately  isolating  the  nitrous  organism  on  the 
surface  of  gelatinous  silica  containing  the  necessary  inorganic 

1  The  saltpetre  beds  of  Chili  and  Peru  are  an  excellent  example  of  the  indus- 
trial application  of  these  facts.  Nitrates  are  there  produced  from  the  faecal 
evacuations  of  sea-fowl  in  such  quantities  as  to  form  an  article  of  commerce. 
A  like  form  of  utilisation  of  the  action  of  these  bacteria  was  once  practiced  on 
the  continent  of  Europe.  Economic  application  is  also  seen  in  the  treatment 
of  sewage  referred  to  elsewhere. 


BACTERIA   IN   THE   SOIL  155 

food.  Professor  Warington,  in  his  lectures  under  the  Lawes 
Agricultural  Trust,  has  described  this  important  germ  as 
follows : 

"  The  organism  as  found  in  suspension  in  a  freshly  nitrified 
solution  consists  largely  of  nearly  spherical  corpuscles,  varying 
extremely  in  size.  The  largest  of  these  corpuscles  barely  reaches 
a  diameter  of  one-thousandth  of  a  millimetre,  and  some  are  so 
minute  as  to  be  hardly  discernible  in  photographs.  The  larger 
ones  are  frequently  not  strictly  circular,  and  are  sometimes  seen 
in  the  act  of  dividing. 

"  Besides  the  form  just  described,  there  is  another,  not  uni- 
versally present  in  solutions,  in  which  the  length  is  considerably 
greater  than  its  breadth.  The  shape  varies,  being  occasionally 
a  regular  oval,  but  sometimes  largest  at  one  end,  and  sometimes 
with  the  ends  truncated.  The  circular  organisms  are  probably 
the  youngest. 

"  This  organism  grows  in  broth,  diluted  milk,  and  other  solu- 
tions without  producing  turbidity.  When  acting  on  ammonia  it 
produces  only  nitrites.  It  is  without  action  on  potassium  nitrite. 
It  is,  in  fact,  the  nitrous  organism  which,  as  we  have  previously 
seen,  may  be  separated  from  soil  by  successive  cultivations  in 
ammonium  carbonate  solution." 

The  elongated  forms  appear  to  be  a  sign  of  arrested 
growth.  Normally  the  organ  is  about  1.8  ^  long,  or  nearly 
three  times  as  long  as  the  nitric  organism.  It  possesses  a 
gelatinous  capsule.  '  The  motile  cells,  stained  by  Loffler's 
method,  are  seen  to  have  a  flagellum  in  the  form  of  a  spiral." 
When  grown  on  silica  the  nitrous  organism  appears  in 
the  same  two  forms — zooglea  and  free  cells — as  when  culti- 
vated in  a  fluid.  It  commences  to  show  growth  in  about 
four  days,  and  is  at  its  maximum  on  about  the  tenth  day. 
Winogradsky  found  that  there  were  considerable  differ- 
ences in  the  morphology  of  the  organism  according  to  the 
soil  from  which  it  was  taken.  One  of  the  Java  soils  he 
investigated  contained  a  nitrous  organism  having  a  spiral 


156  BACTERIA 

flagellum  of  thirty  micromillimetres ;  but  its  movement  was 
slow. 

As  we  have  already  seen,  the  most  astonishing  property 
of  this  organism  is  its  ability  to  grow  and  perform  its  specific 
function  in  solutions  absolutely  devoid  of  organic  matter. 
Some  authorities  hold  that  it  acquires  its  necessary  carbon 
from  carbonic  acid.  The  mode  of  culturing  it  is  as  fol- 
lows: 

To  sterilised  flasks  add  100  cc.  of  a  solution  made  of  one 
gram  of  ammonium  sulphate,  one  gram  of  potassium  sul- 
phate, and  1000  cc.  of  pure  water.  To  this  add  one  gram 
of  basic  magnesium  carbonate  which  has  been  previously 
sterilised  by  boiling.  Now  inoculate  the  flask  with  a  small 
portion  of  the  soil  under  investigation,  and  after  four  or  five 
days  sub-culture  on  the  same  medium  in  fresh  flasks,  and 
let  this  be  repeated  half  a  dozen  times.  Now,  as  this  in- 
organic medium  is  unfavourable  to  ordinary  bacteria  of  soil, 
it  is  clear  that  after  several  sub-cultures  the  nitrous  organism 
will  be  isolated  in  pure  culture. 

Winogradsky  employs  for  culturing  upon  solid  media  a 
mineral  gelatine.  A  solution  of  from  3  to  4  per  cent,  of 
silicic  acid  in  distilled  water  is  placed  in  flasks.  By  the 
addition  of  the  following  salts  to  such  a  solution  gelatinisa- 
tion  occurs: 

(  Ammonium  sulphate 0.4  gram 

(a)  <  Magnesium  sulphate 0.05 

(  Calcium  chloride A  trace 

(  Potassium  phosphate o.i      gram 

(b)  <  Sodium  carbonate 0.6,  0.9 

(  Distilled  water 100  cc. 

The  sulphates  and  chloride  are  mixed  in  50  cc.  of  distilled 
water,  and  the  latter  substance  in  the  remaining  50  cc.  in 
separate  flasks.  After  sterilisation  and  cooling  these  are  all 
mixed  and  added  in  small  quantities  to  the  silicic  acid. 


BACTERIA   IN   THE   SOIL  Itf 

Upon  this  medium  it  is  possible  to  sub-culture  a  pure 
growth  from  the  film  at  the  bottom  of  the  flasks  in  which 
the  nitrous  organism  is  first  isolated. 

The  Nitric  Organism.  It  was  soon  learned  that  the 
nitrous  organism,  even  when  obtainable  in  large  quantities 
and  in  pure  culture,  was  not  able  entirely  to  complete  the 
nitrifying  process.  As  early  as  1881  Professor  Warington 
had  observed  that  some  of  his  cultures,  though  capable  of 
changing  nitrites  into  nitrates,  had  no  power  of  oxidising 
ammonia.  These  he  had  obtained  from  advanced  sub- 
cultures of  the  nitrous  organism,  and  somewhat  later  Wino- 
gradsky  isolated  and  described  this  companion  of  the  nitrous 
organism.  It  develops  freely  in  solutions  to  which  no 
organic  matter  has  been  added ;  indeed,  much  organic  mat- 
ter will  prevent  its  growing.  He  isolated  it  from  soils  from 
various  parts  of  the  world  on  the  following  media : 

Water 1000.0 

Potassium  phosphate i.o 

Magnesium  sulphate 0.5 

Calcium  chloride A  trace 

Sodium  chloride 2.0 

About  20  cc.  of  this  solution  is  placed  in  a  flat-bottom 
flask,  and  a  little  freshly  washed  magnesium  carbonate  is 
added.  The  flask  is  closed  with  cotton  wool,  and  the  whole 
is  sterilised.  To  each  flask  2  cc.  of  a  2  per  cent,  solution 
of  ammonium  sulphate  is  subsequently  added.  The  tem- 
perature for  incubation  is  30°  C.  Winogradsky  concluded 
that  the  oxidation  of  nitrites  to  nitrates  was  brought  about 
by  a  specific  organism  independently  of  the  nitrous  organ- 
ism. He  successfully  isolated  it  in  silica  jelly.  He  believes 
the  organism,  like  its  companion,  derives  its  nutriment 
solely  from  inorganic  matter,  but  this  is  not  finally  estab- 
lished. 

The  form  of   the  nitric  organism   (or   nitromonas,  as   it 


158  BACTERIA 

was  once  termed)  is  allied  to  the  nitrous  organism.  The 
cells  are  elongated,  rarely  oval,  but  sometimes  pear-shaped. 
They  are  more  than  half  a  micromillimetre  in  length,  and 
somewhat  less  in  thickness.  The  cells  have  a  gelatinous 
membrane.  Like  the  other  nitrifying  bacteria,  its  develop- 
ment and  action  are  favoured  by  the  presence  of  the  acid 
carbonates  of  calcium  and  sodium.  Of  the  latter,  six  grams 
per  litre  or  even  a  smaller  quantity  gives  good  results.  The 
sulphate  of  calcium  can  be  used,  but  the  organism  prefers 
the  carbonates.  The  differences  between  these  two  bacteria 
are  small,  with  the  exception  of  their  chemical  action.  The 
nitric  organism  has  no  action  upon  ammonia,  and  the  pres- 
ence of  any  considerable  amount  of  ammonium  carbonate 
hinders  its  development  and  prevents  its  action  on  a  nitrite.1 
We  may  here  summarise  the  general  facts  respecting  nitri- 
fication. Winogradsky  proposes  to  term  the  group  nitro- 
bacteria,  and  to  classify  thus : 

(Nitrosomonas,    containing    at    least    two 
species,    viz.,    the    European    and    the 
Java. 
Nitrosococcus. 
Nitric  organism       =     Nitrobacter. 

Nitrification  occurs  in  two  stages,  each  stage  performed 
by  a  distinct  organism.  By  one  (nitrosomonas)  ammonia  is 
converted  into  nitrite ;  by  the  other  (nitrobacter)  the  nitrite 
is  converted  into  nitrate.2  Both  organisms  are  widely  and 

1  The  course  of  nitrification  may  be  followed  by  means  of  chemical  tests. 
i.  The  disappearance  of  ammonia.     2,   The  appearance  of  nitrite.     3.   Its  dis- 
appearance.    4.  Appearance  of  nitrate. 

2  Professor  Warington,  in  Report  IV.   (p.    526)  of  his  admirable  series  of 
papers  on  the  subject,  draws  attention  to  Mtintz's  criticism  that  the  nitrifying 
organisms  only  oxidise  from  nitrogenous  matter  to  nitrites,  and  not  from  nitrites 
to  nitrates.     Mttntz  held  that  the  conversion  of  nitrite  into  nitrate  is  brought 
about  by  the  joint  action  of  carbonic  acid  and  oxygen.     Professor  Warington's 
experiments,  however,  clearly  illustrate  that  the  production  of  nitrates  from 


NITROUS  ORGANISM 

X  1000 


NITRIC  ORGANISM 

X  looo 


NITROGEN  FIXING  ORGANISM  FROM  SECRETION  OF  ROOT-NODULES 


X  1000 


BACTERIA   IN    THE   SOIL  159 

abundantly  distributed  in  the  superficial  soils.  They  act 
together  and  in  conjunction,  and  for  one  common  purpose. 
They  are  separable  by  employing  favourable  media. 

"  If  we  employ  a  suitable  inorganic  solution  containing  potas- 
sium nitrite,  but  no  ammonia,  we  shall  presently  obtain  the  nitric 
organism  alone,  the  nitrous  organism  feeding  on  ammonia  being 
excluded.  If,  on  the  other  hand,  we  employ  an  ammonium 
carbonate  solution  of  sufficient  strength,  we  have  selected  con- 
ditions very  unfavourable  to  the  growth  of  the  nitric  organism, 
and  a  few  cultivations  leave  the  nitrous  organism  alone  in  pos- 
session of  the  field  "  (Warington). 

A  word  upon  the  natural  distribution  of  these  nitrifying 
bacteria  before  we  leave  them.  They  belong  to  the  soil, 
river  water,  and  sewage.  They  are  also  said  to  be  fre- 
quently present  in  well  water.  From  some  experiments  at 
Rothamsted  it  appears  that  the  organisms  occur  mostly  in 
the  first  twelve  inches,  and  in  subsoils  of  clay  down  to  three 
or  four  feet.  In  sandy  soils  nitrification  may  probably 
occur  at  a  greater  depth.  These  facts  should  be  borne  in 
mind  when  arranging  for  the  purification  of  sewage  by  in- 
termittent filtration. 

We  have  now  given  some  consideration  to  the  chief 
events  in  the  life-cycle  of  nature  depicted  in  the  table. 
There  is  but  one  further  process  in  which  bacteria  play  a 
part,  and  which  requires  some  mention.  It  will  have  been 
noticed  that  at  certain  stages  in  the  cycle  there  is  more  or 
less  appreciable  "  loss  "  of  free  nitrogen.  In  the  process  of 
decomposition  brought  about  by  the  denitrifying  bacteria, 
a  very  considerable  portion  of  the  nitrogen  is  dissipated 

nitrites  in  an  ammoniacal  solution  can  be  determined  by  the  character  of  the 
bacterial  culture  with  which  the  solution  is  seeded,  and  that  in  a  solution  of 
potassium  nitrite  conversion  into  nitrate  can  be  determined  by  the  introduction 
of  the  nitric  organism.  Professor  Warington  still  adheres  to  the  opinion,  in 
favour  of  which  he  has  produced  so  much  evidence,  that  the  formation  of 
nitrates  in  the  soil  is  due  to  the  nitric  organism  which  soil  always  contains. 


l6o  BACTERIA 

into  the  air  in  the  form  of  a  free  gas.  This  is  the  last  stage 
of  all  proteid  decomposition,  so  that  wherever  putrefaction 
is  going  on  there  is  a  continual  "  loss  "  of  an  element  essen- 
tial to  life.  Thus  it  would  appear  at  first  sight  that  the 
sum-total  of  nitrogen  food  must  be  diminishing. 

But  there  are  other  ways  also  in  which  nitrogen  is  being 
set  free.  In  the  ordinary  processes  of  vegetation  there  is  a 
gradual  draining  of  the  soil  and  a  passing  of  nitrogen  into 
the  sea;  the  products  of  decomposition  pass  from  the  soil 
by  this  drainage,  and  are  "  lost  "  as  far  as  the  soil  is  con- 
cerned. Many  of  the  methods  of  sewage  disposal  are  in 
reality  depriving  the  land  of  the  return  of  nitrogen  which  is 
its  necessity.  Again,  nitrogen  is  freed  in  explosions  of  gun- 
powder, nitroglycerine,  and  dynamite,  for  whatever  purpose 
they  are  used.  Hence  the  great  putrefactive  "  loss  "  of 
nitrogen,  with  its  subsidiary  losses,  contributes  to  reduce 
this  essential  element  of  all  life,  and  if  there  were  no  method 
of  bringing  it  back  again  to  the  soil,  it  would  seem  that 
plant  life,  and  therefore  animal  life,  would  speedily  terminate. 

It  is  at  this  juncture,  and  to  perform  this  vital  function, 
that  the  nitrogen-fixing  bacteria  play  their  wonderful  part : 
they  bring  back  the  free  nitrogen  and  fix  it  in  the  soil.  Ex- 
cepting a  small  quantity  of  combined  nitrogen  coming  down 
in  rain  and  in  minor  aqueous  deposits  from  the  atmosphere, 
the  great  source  of  the  nitrogen  of  vegetation  is  the  store 
in  the  soil  and  subsoil,  whether  derived  from  previous  ac- 
cumulations or  from  recent  supplies  by  manure. 

Sir  William  Crookes  has  recently  1  pointed  out  the  vast 
importance  of  using  all  the  available  nitrogen  in  the  service 
of  wheat  production.  The  distillation  of  coal  in  the  process 
of  gas-making  yields  a  certain  amount  of  its  nitrogen  in  the 
form  of  sulphate  of  ammonia,  and  this,  like  other  nitro- 
genous manures,  might  be  used  to  give  back  to  the  soil 

1  British  Association  for  the  Advancement  of  Science,  Bristol,  1898,  Presi- 
dential Address. 


BACTERIA   IN   THE   SOIL  l6l 

some  of  the  nitrogen  drained  from  it.  But  such  manuring 
cannot  keep  pace,  according  to  Sir  W.  Crookes,  with  the 
present  loss  of  fixed  nitrogen  from  the  soil.  We  have  al- 
ready referred  to  several  ways  in  which  "  loss  "  of  nitrogen 
occurs.  To  these  may  well  be  added  the  enormous  loss  oc- 
curring in  the  waste  of  sewage  when  it  is  passed  into  the 
sea.  As  the  President  of  the  British  Association  pointed 
out,1  the  more  widely  this  wasteful  system  is  extended, 
recklessly  returning  to  the  sea  what  we  have  taken  from  the 
land,  the  more  surely  and  quickly  will  the  finite  stocks  of 
nitrogen,  locked  up  in  the  soils  of  the  world,  become  ex- 
hausted. Let  us  remember  that  the  plant  creates  nothing 
in  this  direction ;  there  is  nothing  in  wheat  which  is  not 
absorbed  from  the  soil,  and  unless  the  abstracted  nitrogen 
is  returned  to  the  soil,  its  fertility  must  be  ultimately  ex- 
hausted. When  we  apply  to  the  land  sodium  nitrate,  sul- 
phate of  ammonia,  guano,  and  similar  manurial  substances, 
we  are  drawing  on  the  earth's  capital,  and  our  drafts  will 
not  be  prepetually  responded  to.2  We  know  that  a  virgin 
soil  cropped  for  several  years  loses  its  productive  powers, 
and  without  artificial  aid  becomes  unfertile.  For  example, 
through  this  exhaustion  forty  bushels  of  wheat  per  acre 
have  dwindled  to  seven.  Rotation  of  crops  is  an  attempt 
to  meet  the  problem,  and  the  four-course  rotation  of  turnips, 
barley,  clover,  and  wheat  witnesses  to  the  fact  that  practice 
has  been  ahead  of  science  in  this  matter. 

The  store  of  nitrogen  in  the  atmosphere  is  practically 
unlimited,  but  it  is  fixed  and  rendered  assimilable  only  by 
cosmic  processes  of  extreme  slowness.  We  may  shortly 
glance  at  these,  for  it  is  upon  these  processes,  plus  a  return 

*  British  Association  for  the  Advancement  of  Science,  Bristol,  1898,  Presi- 
dential Address. 

2  Sir  John  Lawes  and  Sir  Henry  Gilbert  (Times,  December  2,  1898),  have 
pointed  out  that  the  addition  of  nitrates  only  would  be  of  no  permanent  use  to 
the  wheat  crop.  They  rely  upon  thorough  tillage  and  proper  rotation  of  crops 
as  the  means  of  improving  the  nitrogen  value  of  the  soil. 


1 62  BACTERIA 

to  the  soil  of  sewage,  that  we  must  depend  in  the  future  for 
storing  nitrogen  as  nitrates. 

1.  Some  combined    nitrogen  is  absorbed  by  the  soil  or 
plant  from  the  air,  for  example,  fungi,  lichens,  and  some 
algae,  and  the  absorption  is  in  the  form  of  ammonia  and 
nitric  acid.     This  is  admittedly  a  small  quantity. 

2.  Some  free  nitrogen  is   fixed  within  the  soil  by  the 
agency  of  porous  and  alkaline  bodies. 

3.  Some,  again,  may  be  assimilated  by  the  higher  chlo- 
rophyllous   plants   themselves,    independently  of   bacteria 
(Frank). 

4.  Electricity  fixes,  and  may  in  the  future  be  made  to  fix 
more,  nitrogen.     If  a  strong  inductive  current  be  passed  be- 
tween terminals,  the  nitrogen  from  the  air  enters  into  com- 
bination with  the  oxygen,  producing  nitrous  and  nitric  acids. 

5.  Abundant  evidence  has  now  been  produced  in  support 
of  the  fact  that  there  is  considerable  fixation  by  means  of 
bacteria. 

Bacterial  life  in  several  ways  is  able  to  reclaim  from  the 
atmosphere  this  free  nitrogen,  which  would  otherwise  be 
lost.  The  first  method  to  which  reference  may  be  made  is 
that  involving  symbiosis.  This  term  signifies  "  a  living 
together"  of  two  different  forms  of  life,  generally  for  a 
specific  purpose.  It  may  be  to  mutual  advantage',  a  living 
for  one  another,  or  it-may  be,  by  means  of  an  interchange 
of  metabolism  or  products,  finally  to  produce  or  obtain  some 
remote  chemical  result.  It  is  convenient  to  restrict  the  term 
symbiosis  to  complementary  partnerships  such  as  exist  be- 
tween algoid  and  fungoid  elements  in  lichens,  or  between 
unicellular  algae  and  Radiolarians,1  or  between  bacteria  and 
higher  plants.  The  partnerships  between  hermit  crabs  and 
sea-anemones  and  the  like  are  sometimes  defined  by  the  term 
commensalism  (joint  diet).  Symbiosis  and  commensaHsm 
must  be  distinguished  from  parasitism,  which  indicates  that 

1  Geddes,  Nature,  xxv.,  1882. 


BACTERIA    IN    THE   SOIL 


all  the  advantage  is  on  the  side  of  the  parasite,  and  nothing 
but  loss  on  the  side  of  the  host.  The  distinction  between 
symbiosis  and  commesalism  cannot  be  rigid,  but  between 
these  conditions  which  are  advantageous  to  the  partners  and 
parasitism,  there  is  an  obvious  and  radical  difference.  As- 
sociation of  organisms  together  for  increase  of  virulence  and 
function  should  be  distinguished  from  symbiosis,  and  mere 
existence  of  two  or  more  species 
of  bacteria  in  one  medium  is 
not,  of  course,  symbiosis.  Most 
frequently  such  a  condition 
would  result  in  injury  and  the 
subsequent  death  of  the  weaker 
partner,  an  effect  precisely  op- 
posite to  that  defined  by  this 
term. 

The  example  of  bacteriologi- 
cal symbiosis  with  which  we  are 
concerned  here  is  that  partner- 
ship between  bacteria  and  some 
of  the  higher  plants  (Legumin- 
osce)  for  the  purpose  of  fixing 
nitrogen  in  the  plant  and  in  the 
surrounding  soil. 

The  Nitrogen- fixing  Bacteria, 
the  third  group  of  micro-organ- 
isms connected  with  the  soil, 
exist  in  groups  and  colonies 
situated  inside  the  nodules  ap- 
pearing, under  certain  circum- 
stances, on  the  rootlets  of  the 
pea,  bean,  and  other  Legumin-  RoQTLsn  OF  PEA 
osce.  It  was  Hellriegel  and 
Wilfarth  who  first  pointed  out  that,  although  the  higher 
chlorophyllous  plants  could  not  directly  obtain  or  utilise  free 


164  BACTERIA 

nitrogen,  some  of  them  at  any  rate  could  acquire  nitrogen 
brought  into  combination  under  the  influence  of  bacteria. 
Hellriegel  found  that  the  gramineous,  polygonaceous,  cruci- 
ferous, and  other  orders  depended  upon  combined  nitrogen 
supplied  within  the  soil,  but  that  the  Leguminoscz  did  not 
depend  entirely  upon  such  supplies. 

It  was  observed  that  in  a  series  of  pots  of  peas  to  which 
no  nitrogen  was  added  most  of  the  plants  were  apparently 
limited  in  their  growth  by  the  amount  of  nitrogen  locked 
up  in  the  seed.  Here  and  there,  however,  a  plant,  under 
apparently  the  same  circumstances,  grew  luxuriantly  and 
possessed  on  its  rootlets  abundant  nodules.  The  experi- 
ments of  Sir  John  Lawes  and  Sir  Henry  Gilbert  at  Rotham- 
sted '  demonstrated  further  that  under  the  influence  of 
suitable  microbe-seeding  of  the  soil  in  which  Leguminosce 
were  planted  there  is  nodule  formation  on  the  roots,  and 
coincidentally  increased  growth  and  gain  of  nitrogen  beyond 
that  supplied  either  in  the  soil  or  in  the  seed  as  combined 
nitrogen.  Presumably  this  is  due  to  the  fixation,  in  some 
way,  of  free  nitrogen.  Nobbe  proved  the  gain  of  nitrogen 
by  non-leguminous  plants  (Eloeagnus,  etc.)  when  these  grow 
root  nodules  containing  bacteria,  but  to  all  appearances 
bacteria  differing  morphologically  from  the  Bacillus  radi- 
cicola  of  the  leguminous  plants. 

These  facts  being  established,  the  question  naturally 
arises,  How  is  the  fixation  of  nitrogen  to  be  explained,  and 
by  what  species  of  bacteria  is  it  performed  ?  In  the  first 
place,  these  matters  are  simplified  by  the  fact  that  there  is 
very  little  fixation  indeed  by  bacteria  in  the  soil  apart  from 
symbiosis  with  higher  plants.  Hence  we  have  to  deal 
mainly  with  the  work  of  bacteria  in  the  higher  plant.  Sir 
Henry  Gilbert  concludes  2  that  the  alternative  explanations 

JSir  Henry  Gilbert,  F.R.S.,  The  Lawes  Agricultural  Trust  Lectures,  1893, 
p.  129. 

2  Ibid.,  p.  140. 


Cellular 
sheath  of 
Rootlet 
forming 
capsule 
of  nodule. 


NITROGEN  FIXING  BACTERIA  IN  SITU  IN  NODULE  ON  ROOTLET  OF  PEA 

X  400 


NITROGEN  FIXING  BACTERIA  IN  SITU 

IN  ROOT-NODULE  OF  PEA 

(Section  of  Nodule) 

X  500 


NITROGEN  FIXING  BACTERIA  IN  SITU 
IN  ROOT-NODULE  OF  PEA 

(Section  of  Nodule) 
X  600 


BACTERIA   IN   THE   SOIL  165 

of  the  fixation  of  free  nitrogen  in  the  growth  of  Leguminosce 
seem  to  be: 

"  i.  That  under  the  conditions  of  symbiosis  the  plant  is  en- 
abled to  fix  the  free  nitrogen  of  the  atmosphere  by  its  leaves  ; 

"  2.  That  the  nodule  organisms  become  distributed  within  the 
soil  and  there  fix  free  nitrogen,  the  resulting  nitrogenous  com- 
pounds becoming  available  as  a  source  of  nitrogen  to  the  roots 
of  the  higher  plant  ; 

"  3.  That  free  nitrogen  is  fixed  in  the  course  of  the  develop- 
ment of  the  organisms  within  the  nodules,  and  that  the  resulting 
nitrogenous  compounds  are  absorbed  and  utilized  by  the  host." 
"  Certainly,"  he  adds,  "  the  balance  of  evidence  at  present  at 
command  is  much  in  favour  of  the  third  mode  of  explanation." 

If  this  is  finally  proved  to  be  the  case,  it  will  furnish  another 
excellent  example  of  the  power  existing  in  bacteria  of  as- 
similating an  elementary  substance. 

Most  authorities  would  agree  that  all  absorption  of  free 
nitrogen,  if  by  means  of  bacteria,  must  be  through  the  roots. 
As  a  matter  of  fact,  legumes,  especially  when  young,  use 
nitrogen,  like  all  other  plants,  derived  from  the  soil.  It 
has  been  pointed  out  that,  unless  the  soil  is  somewhat  poor 
in  nitrogen,  there  appears  to  be  but  little  assimilation  of 
free  nitrogen  and  but  a  poor  development  of  root  nodules.1 
The  free  nitrogen  made  use  of  by  the  micro-organism  is  in 
the  air  contained  in  the  interstices  of  the  soil.  For  in  all 
soils,  but  especially  in  well-drained  and  light  soils,  there  is 
a  large  quantity  of  air.  Although  it  is  not  known  how  the 
micro-organisms  in  legumes  utilise  free  nitrogen  and  convert 
it  into  organic  compounds  in  the  tissues  of  the  rootlet  or 
plant,  it  is  known  that  such  nitrogen  compounds  migrate 
into  the  stem  and  leaves,  and  so  make  the  roots  really  poorer 

1  This  has  been  denied  recently  in  the  official  report  by  the  chemist  of  the 
Experimental  Farm  to  the  Minister  of  Agriculture  at  Ottawa  (Report,  1896, 
p.  200). 


1 66  BACl^ERIA 

in  nitrogen  than  the  foliage.  But  the  ratio  is  a  fluctuating 
one,  depending  chiefly  on  the  stage  of  growth  or  maturity 
of  the  plant. 

If  the  nodules  from  the  rootlets  of  Leguminosce  be  ex- 
amined, the  nitrogen-fixing  bacteria  can  be  readily  seen. 
The  writer  has  isolated  these  and  grown  them  in  pure  cult- 
ure as  follows:  The  nodules  are  removed,  if  possible  at  an 
early  stage  in  their  growth,  and  placed  for  a  few  minutes  in 
a  steam  steriliser.  This  is  advisable  in  order  to  remove  the 
various  extraneous  organisms  attached  to  the  outer  covering 
of  the  nodule.  They  may  then  be  washed  in  antiseptic 
solution,  and  their  capsules  softened  by  soaking.  When 
opened  with  a  sterile  knife,  thick  creamy  matter  exudes. 
On  microscopic  examination  this  is  found  to  be  densely 
crowded  with  small  round-ended  bacilli  or  oval  bodies, 
known  as  bacteroids.  By  a  simple  process  of  hardening  and 
using  the  microtome,  excellent  sections  of  the  nodules  can 
be  obtained  which  show  these  bacteria  in  situ.  In  the  cen- 
tral parts  of  the  section  may  be  seen  densely  crowded  colo- 
nies of  the  bacteria,  which  in  some  cases  invade  the  cellular 
capsule  of  the  nodule  derived  from  the  rootlet.  Aerobic  and 
anaerobic  pure  cultures  of  these  bacteria  were  made.  In 
some  cases  these  cultures  very  closely  resembled  the  feathery 
growth  of  the  bacillus  of  anthrax. 

4.  The  Saprophytic  Bacteria  in  Soil.  This  group  of 
micro-organisms  is  by  far  the  most  abundant  as  regards 
number.  They  live  on  the  dead  organic  matter  of  the  soil, 
and  their  function  appears  to  be  to  break  it  down  into 
simpler  constitution.  Specialisation  is  probably  progressing 
among  them,  for  their  name  is  legion,  and  the  struggle  for 
existence  keen.  After  we  have  eliminated  the  economic 
bacteria,  most  of  which  are  obviously  saprophytes,  the 
group  is  greatly  reduced.  It  is  also  needless  to  add  that  of 
the  remnant  little  beyond  morphology  is  known,  for  as  their 
function  is  learned  they  are  classified  otherwise.  It  is  prob- 


BACTERIA    IN    THE   SOIL  167 

able,  as  suggested,  that  many  of  the  species  of  common 
saprophytes  normally  existent  in  the  soil  act  as  auxiliary 
agents  to  denitrification  and  putrefaction.  At  present  we 
fear  they  are  disregarded  in  equal  measure,  and  for  the  same 
reasons,  as  the  common  water  bacteria.  An  excess  of  either, 
in  soil  or  water,  is  not  of  itself  injurious  as  far  as  we  know; 
indeed,  it  is  probably  just  the  reverse.  It  is,  however,  fre- 
quently an  index  of  value  as  to  the  amount  and  sometimes 
condition  of  the  contained  organic  matter.  The  remarks 
made  when  considering  water  bacteria  apply  here  also,  viz., 
that  an  excess  of  saprophytes  acts  not  only  as  index  of  in- 
crease of  organic  matter,  but  as  at  first  auxiliary,  and  then 
detrimental,  to  pathogenic  organisms.  It  will  require  ac- 
curate knowledge  of  soil  bacteria  generally  to  be  able  to  say 
which  saprophytic  germs,  if  any,  have  no  definite  function 
beyond  their  own  existence.  It  may  be  doubted  whether 
the  stern  behests  of  nature  permit  of  such  organisms.  How- 
ever that  may  be,  we  may  feel  confident,  though  at  present 
there  are  many  common  bacteria  in  soil,  as  also  in  water, 
the  life  object  of  which  is  not  ascertained,  that  as  knowledge 
increases  and  becomes  more  accurate  this  special  provisional 
group  will  become  gradually  absorbed  into  other  groups 
having  a  part  in  the  economy  of  nature,  or  in  the  production 
of  disease.  At  present  the  decomposition,  denitrifying, 
nitrifying,1  and  nitrogen-fixing  organisms  are  the  only  sap- 
rophytes which  have  been  rescued  from  the  oblivion  of  ages, 
and  brought  more  or  less  into  daylight.  It  is  but  our  lack 
of  knowledge  which  requires  the  present  division  of  sap- 
rophytes whose  business  and  place  in  the  world  is  unknown. 
5.  The  Pathogenic  Organisms  found  in  Soil.  In  addition 
to  these  saprophytes  and  the  economic  bacteria,  there  are, 
as  is  now  well  known,  some  disease-producing  bacteria  find- 

1  It  has  already  been  pointed  out  that  the  nitrifying  bacteria,  though  able  to 
live  on  organic  matter,  do  not  require  such  either  for  existence  or  for  the  per- 
formance of  their  function. 


1 68  BACTERIA 

ing  their  nidus  in  ordinary  soil.  The  three  chief  members 
of  this  group  are  the  bacillus  of  Tetanus  (lockjaw),  the 
bacillus  of  Quarter  Evil,  and  the  bacillus  of  Malignant 
CEdema. 

Tetanus.  The  pathology  of  this  terrible  disease  has 
during  recent  years  been  considerably  elucidated.  It  was 
the  custom  to  look  upon  it  as  "  spontaneous,"  and  arising 
no  one  knew  how ;  now,  however,  after  the  experiments  of 
Sternberg  and  Nicolaier,  the  disease  is  known  to  be  due  to 
a  micro-organism  common  in  the  soil  of  certain  localities, 
existing  there  either  as  a  bacillus  or  in  a  resting  stage  of 
spores.  Fortunately  Tetanus  is  comparatively  rare,  and 
one  of  the  peculiar  biological  characteristics  of  the  bacillus  is 
that  it  grows  only  in  the  absence  of  oxygen.  This  fact  con- 
tributed not  a  little  to  the  difficulties  which  were  met  with 
in  securing  its  isolation. 

Tetanus  occurs  in  man  and  horses  most  commonly,  though 
it  may  affect  other  animals.  There  is  usually  a  wound, 
often  an  insignificant  one,  which  may  occur  in  any  part  of 
the  body.  The  popular  idea  that  a  severe  cut  between  the 
thumb  and  the  index  finger  leads  to  tetanus  is  without 
scientific  foundation.  As  a  matter  of  fact,  the  wound  is 
nearly  always  on  one  or  other  of  the  limbs,  and  is  infected 
simply  because  they  come  more  into  contact  with  soil  and 
dust  than  does  the  trunk.  It  is  not  the  locality  of  the 
wound  nor  its  size  that  affects  the  disease.  A  cut  with  a 
dirty  knife,  a  gash  in  the  foot  from  the  prong  of  a  gardener's 
fork,  the  bite  of  an  insect,  or  even  the  prick  of  a  thorn  have 
before  now  set  up  tetanus.  Wounds  which  are  jagged,  and 
occurring  in  absorptive  tissues,  are  those  most  fitted  to  allow 
the  entrance  of  the  bacillus.  The  wound  forms  a  local  man- 
ufactory, so  to  speak,  of  the  bacillus  and  its  secreted  poisons  ; 
the  bacillus  always  remains  in  the  wound,  but  the  toxins  may 
pass  throughout  the  body,  and  are  especially  absorbed  by 
the  cells  of  the  central  nervous  system,  and  thus  give  rise 


BACTERIA    IN    THE   SOIL  169 

to  the  spasms  which  characterise  the  disease.  Suppuration 
generally  occurs  in  the  wound,  and  in  the  pus  thus  produced 
may  be  found  a  great  variety  of  bacteria,  as  well  as  the 
specific  agent  itself.  After  a  few  days  or,  it  may  be,  as 
much  as  a  fortnight,  when  the  primary  wound  may  be 
almost  forgotten,  general  symptoms  occur.  Their  appear- 
ance is  often  the  first  sign  of  the  disease.  Stiffness  of  the 
neck  and  facial  muscles,  including  the  muscles  of  the  jaw, 
is  the  most  prominent  sign.  This  is  rapidly  followed  by 
spasms  and  local  convulsions,  which,  when  affecting  the  re- 
spiratory or  alimentary  tract,  may  cause  a  fatal  result. 
Fever  and  increased  rate  of  pulse  and  respiration  are  further 
signs  of  the  disease  becoming  general.  After  death,  which 
results  in  the  majority  of  cases,  there  is  very  little  to  show 
the  cause  of  fatality.  The  wound  is  observable,  and  patches 
of  congestion  may  be  found  on  different  parts  of  the  nervous 
system,  particularly  the  medulla  (grey  matter),  pons,  and 
even  cerebellum.  Evidence  has  recently  been  forthcoming 
at  the  Pasteur  Institute  to  support  the  theory  that  tetanus 
is  a  nervous  disease,  more  or  less  allied  to  rabies,  and  is  best 
treated  by  intra-cerebral  injection  of  antitoxin,  which  then 
has  an  opportunity  of  opposing  the  toxins  at  their  favourite 
site.  (Roux  and  Borrel.) 

In  the  wound  the  bacillus  is  present  in  large  numbers, 
but  mixed  up  with  a  great  variety  of  suppurative  bacteria 
and  extraneous  organisms.  It  is  in  the  form  of  a  straight 
short  rod  with  rounded  ends,  occurring  singly  or  in  pairs  of 
threads,  and  slightly  motile.  It  has  been  pointed  out  that 
by  special  methods  of  staining,  flagella  may  be  demon- 
strated.1 These  are  both  lateral  and  terminal,  thin  and 
thick,  and  are  shed  previously  to  sporulation.  Branching 
also  has  been  described.  Indeed,  it  would  appear  that, 
like  the  bacillus  of  tubercle,  this  organism  has  various  pleo- 
morphic  forms.  Next  to  the  ordinary  bacillus,  filamentous 

1  Lehmann  and  Neumann,  p.  3*05. 


I7O  BACTERIA 

forms  predominate,  particularly  so  in  old  cultures.  Clubbed 
forms,  not  unlike  the  bacillus  of  diphtheria,  may  often  be 
seen  from  agar  cultures.  Without  doubt  the  most  peculiar 
characteristic  of  this  bacillus  is  its  sporulation.  The  well- 
formed  round  spores  occur  readily  at  incubation  temperature. 
They  occupy  a  position  at  one  or  other  pole  of  the  bacillus, 
and  have  a  diameter  considerably  greater  than  the  rod. 
Thus  the  well-known  "  drumstick  "  form  is  produced.  In 
practice  the  spores  occur  freely  in  the  medium  and  in  micro- 


"1  V' 

'.    •  *  '  '     ' 


•^ 

'^     * 

2  ^ 

BACILLUS  OF  TETANUS 

scopical  preparation.  Like  other  spores,  they  are  extremely 
resistant  to  heat,  desiccation,  and  antiseptics.  They  can 
resist  boiling  for  several  minutes. 

As  we  have  seen,  this  bacillus  is  a  strict  anaerobe,  grow- 
ing only  in  the  absence  of  oxygen.  The  favourable  temper- 
ature is  37°  C. ,  and  it  will  only  grow  very  slowly  at  or  below 
room  temperature. 

An  excellent  culture  is  generally  obtainable  in  glucose 
gelatine.  The  growth  occurs,  of  course,  only  in  the  depth 
of  the  medium,  and  appears  as  fine  threads  passing  horizon- 
tally outwards  from  the  track  of  the  needle.  At  the  top 
and  bottom  of  the  growth  these  fibrils  are  shorter  than  at 
the  middle  or  somewhat  below  the  middle.  For  extraction 
of  the  soluble  products  of  the  bacillus  glucose  broth  may  be 
used. 


BACTERIA    IN   THE   SOIL  1 71 

In  some  countries,  and  in  certain  localities,  the  bacillus  of 
tetanus  is  a  very  common  habitant  of  the  soil,  and  when  one 
thinks  how  frequently  wounds  must  be  more  or  less  con- 
taminated with  such  soil,  the  question  naturally  arises,  How 
is  it  that  the  disease  is,  fortunately,  so  rare  ?  Probably  we 
must  look  to  the  advance  of  bacteriological  science  to  answer 
this  and  similar  questions  at  all  adequately.  Much  has  re- 
cently been  done  in  Paris  and  elsewhere  to  emphasise  the 
relation  which  other  organisms  have  to  such  bacteria  as 
those  of  typhoid  and  tetanus.  When  considering  typhoid, 
we  saw  that  in  addition  to  the  presence  of  the  specific  germ 
other  conditions  were  requisite  before  the  disease  actually 
occurred.  So  in  tetanus,  Kitasato  and  others  have  pointed 
out  that  the  presence  of  certain  other  bacteria,  or  of  some 
foreign  body,  is  necessary  to  the  production  of  the  disease. 
The  common  organisms  of  suppuration  are  particularly  ac- 
cused of  increasing  the  virulence  of  the  bacillus  of  tetanus. 
How  these  auxiliary  organisms  perform  this  function  has 
not  been  fully  elucidated.  Probably,  however,  it  is  by 
damaging  the  tissues  and  weakening  their  resistance  to  such 
a  degree  as  to  afford  a  favourable  multiplying  ground  for  the 
tetanus.  It  is  right  to  state  that  some  authorities  hold  that 
they  act  by  using  up  the  surrounding  oxygen,  and  so  favour- 
ing the  growth  of  tetanus. 

Quarter  Evil  (or  symptomatic  anthrax)  is  a  disease  of 
animals,  produced  in  a  manner  analogous  to  tetanus.  It  is 
characterised  by  a  rapidly  increasing  swelling  of  the  upper 
parts  of  the  thigh,  sacrum,  etc.,  which,  beginning  locally, 
may  attain  to  extraordinary  size  and  extent.  It  assumes  a 
dark  colour,  and  crackles  on  being  touched.  There  is  high 
temperature,  and  secondary  motor  and  functional  disturb- 
ances. The  disease  ends  fatally  in  two  or  three  days. 

Slight  injuries  to  the  surface  of  the  skin  or  mucous  mem- 
brane are  sufficient  for  the  introduction  of  the  causal  bacil- 
lus. This  organism  is,  like  tetanus,  an  anaerobe,  existing 


1^2  BACTERIA 

in  the  superficial  layers  of  the  soil.  From  its  habitat  it 
readily  gains  entrance  to  animal  tissues.  It  has  spores,  but 
though  they  are  of  greater  diameter  than  the  bacillus  itself 
they  are  not  absolutely  terminal.  Hence  they  merely  swell 
out  the  capsule  of  the  bacillus,  and  produce  a  club-shaped 
rod.  They  form  gas  while  growing  in  the  tissues  and  in 
artificial  culture.  External  physical  conditons  have  little 
effect  upon  this  bacillus,  and  the  dried  and  even  buried 
flesh  retains  infection  for  a  very  long  period  of  time. 


B.  OF  SYMPTOMATIC  ANTHRAX  B.  OF  MALIGNANT  CEDEMA 

The  third  disease-producing  microbe  found  naturally  in 
soil  is  that  which  produces  the  disease  known  as  Malignant 
CEdema.  Pasteur  called  this  gangrenous  septicaemia.  Unlike 
quarter  evil,  malignant  oedema  may  occur  in  man  in  cases 
where  wounds  have  become  septic.  Animals  become  inocu- 
lated with  this  bacillus  from  the  surface  of  soil,  straw-dust, 
upper  layers  of  garden-earth,  or  decomposing  animal  and 
vegetable  matter. 

The  bacillus  occurs  in  the  blood  and  tissues  as  a  long 
thread,  composed  of  slender  segments  of  irregular  length. 
It  is  motile  and  anaerobic.  The  spores  are  larger  than  the 
diameter  of  the  bacillus,  and  the  organism  produces  gas;  so 
much  is  this  the  case  in  artificial  culture,  that  the  medium 
itself  is  frequently  split  up. 


BACTERIA    IN   THE   SOIL  173 

Both  malignant  oedema  and  symptomatic  anthrax  are 
similar  in  some  respects  to  anthrax  itself.  There  are,  how- 
ever, a  number  of  points  for  differential  diagnosis.  The 
enlargement  of  the  spleen,  the  non-motility  of  the  bacillus, 
the  enormous  numbers  of  bacilli  throughout  the  body,  the 
square  ends,  equal  inter-bacillary  spaces,  aerobic  growth, 
and  characteristic  staining  afford  ample  evidence  of  anthrax. 

The  Relation  of  Soil  generally  to  certain  Bacterial  Diseases. 
Recent  investigations  have,  in  effect,  considerably  added 
to  our  knowledge  of  pathogenic  germs  in  soil ;  and  whilst 
the  three  species  enumerated  above  are  still  considered  as 
types  normally  present  in  soil,  it  must  not  be  forgotten  that 
other  virulent  disease  producers  either  live  in  the  soil  or  are 
greatly  influenced  by  its  conditions. 

Frankel  and  Pasteur  have  both  demonstrated  the  possible 
presence  of  anthrax.  Frankel  maintained  that  it  could  not 
live  there  long,  and  at  ten  feet  below  the  surface  no  growth 
occurred.  This  may  have  been  due  to  the  low  temperature 
of  such  a  depth.  Pasteur  held  that  earthworms  are  respon- 
sible for  conveying  the  spores  of  anthrax  from  buried  car- 
casses to  the  surface,  and  thus  bringing  about  reinfection. 
Cholera,  too,  has  been  successfully  grown  in  soil,  except 
during  winter.  The  presence  of  common  saprophytes  in 
the  soil  is  prejudicial  to  the  development  of  the  cholera 
spirillum,  and  under  ordinary  circumstances  it  succumbs  in 
the  struggle  for  existence.  From  experiments  recently 
conducted  for  the  Local  Government  Board  by  Dr.  Sidney 
Martin,  evidence  is  forthcoming  in  support  of  the  view  that 
the  bacillus  of  typhoid  can  live  in  certain  soils.  Samples  of 
soil  polluted  with  organic  matter  formed  a  favourable  en- 
vironment for  living  bacilli  of  typhoid  for  456  days,  whereas 
in  sterilised  soil,  without  organic  matter,  these  organisms 
lived  only  twenty-three  days.  Tubercle  also  has  been  kept 
alive  for  several  weeks  in  soil. 

In  passing,  a  single  remark  may  be  made  in  relation  to 


174  BACTERIA 

the  long  periods  during  which  bacteria  can  retain  vitality  in 
soil.  Farm  soils  have,  as  is  well  known,  been  contaminated 
with  anthrax  in  the  late  summer  or  autumn,  and  have  re- 
tained the  infectious  virus  till  the  following  spring,  and  it 
has  even  then  cropped  up  again  in  the  hay  of  the  next 
season.  In  1881  Miquel  took  some  samples  of  soil  at  a 
depth  of  ten  inches,  containing  six  and  a  half  million  bac- 
teria per  gram.  After  drying  for  two  days  at  30°  C.,  the 
dust  was  placed  in  hermetically  sealed  tubes,  which  were 
put  aside  in  a  dark  corner  of  the  laboratory  for  sixteen 
years.  Upon  re-examination  it  is  reported  that  more  than 
three  million  germs  per  gram  were  still  found,  amongst 
them  the  specific  bacillus  of  tetanus.  Whether  or  not  there 
is  any  fallacy  in  these  actual  figures,  there  is  abundant  evi- 
dence in  support  of  the  fact  that  bacteria,  non-pathogenic 
and  pathogenic,  can  and  do  retain  their  vitality,  and  some- 
times even  their  virulence,  for  almost  incredibly  long 
periods  of  time. 

It  is  now  some  years  since  Sir  George  Buchanan,  for  the 
English  Local  Government  Board,  and  Dr.  Bowditch,  for  the 
United  States,  formulated  the  view  that  there  is  an  intimate 
relationship  between  dampness  of  soil  and  the  bacterial  dis- 
ease of  Consumption  (tuberculosis  of  the  lungs).  The  mat- 
ter was  left  at  that  time  sub  judice,  but  the  conclusion  has 
been  drawn,  and  surely  a  legitimate  one,  that  the  dampness 
of  the  soil  acted  injuriously  in  one  of  two  ways.  It  either 
lowered  the  vitality  of  the  tissues  of  the  individual,  and  so 
increased  his  susceptibility  to  the  disease,  or  in  some  way 
unknown  favoured  the  life  and  virulence  of  the  bacillus. 
That  is  one  fact.  Secondly,  Pettenkofer  traced  a  definite 
relationship  between  the  rise  and  fall  of  the  ground  water 
with  pollution  of  the  soil  and  enteric  (typhoid)  fever.1  A 

1  The  conditions  requisite  for  an  outbreak  of  enteric  fever  were,  according 
to  Pettenkofer,  (a)  a  rapid  fall  (after  a  rise)  in  the  ground  water,  (l>)  pollution 
of  the  soil  with  animal  impurities,  (c)  a  certain  earth  temperature,  and  lastly 


BACTERIA    IN    THE   SOIL  175 

third  series  of  investigations  concluded  in  the  same  direc- 
tion, viz.,  the  researches  of  Dr.  Ballard  respecting  summer 
diarrhoea.  This,  it  is  generally  held,  is  a  bacterial  disease, 
although  no  single  specific  germ  has  been  isolated  as  its 
cause.  Ballard  demonstrated  that  the  summer  rise  of  diar- 
rhoea mortality  does  not  commence  until  the  mean  temper- 
ature of  the  soil,  recorded  by  the  four-foot  thermometer, 
has  attained  56.4°  F.,  and  the  decline  of  such  diarrhoea 
coincides  more  or  less  precisely  with  the  fall  in  soil  temper- 
ature. This  temperature  (56.4°  F.)  is,  therefore,  considered 
as  the  "  critical  "  four-foot  earth  temperature,  that  is  to 
say,  the  temperature  at  which  certain  changes  (putrefactive, 
bacterial,  etc.)  take  place  in  the  pores  of  the  earth,  with  the 
consequent  development  of  the  diarrhceal  poison. 

After  a  very  elaborate  and  prolonged  investigation  on  be- 
half of  the  Local  Government  Board,  Dr.  Ballard  formulates 
the  causes  of  diarrhoea  in  the  following  conclusions: l 

(a)  The  essential  cause  of  diarrhoea  resides  ordinarily  in 
the  superficial  layers  of  the  earth,  where  it  is  intimately  as- 
sociated with  the  life  processes  of  some  micro-organism  not 
yet  detected  or  isolated. 

(&)  That  the  vital  manifestations  of  such  organism  are 
dependent,  among  other  things,  perhaps  principally  upon 
conditions  of  season  and  the  presence  of  dead^  organic  mat- 
ter, which  is  its  pabulum. 

(c)  That  on  occasion  such  micro-organism  is  capable  of 
getting  abroad  from  its  primary  habitat,  the  earth,  and 
having  become  air-borne,  obtains  opportunity  for  fastening 
on  non-living  organic  material,  and  of  using  such  organic 


(d)  a  specific  micro-organism  in  the  soil.  These  four  conditions  have  not,  par- 
ticularly in  England,  always  been  fulfilled  preparatory  to  an  epidemic  of 
typhoid.  Yet  the  observations  necessary  for  these  deductions  were  a  definite 
step  in  advance  of  mere  dampness  of  soil. 

1  Supplement  to  the  Report  of  the  Medical  Officer  of  the  Local  Government 
Board,  1887,  p.  7. 


176  BACTERIA 

matter  both  as  nidus  and  as  pabulum  in  undergoing  various 
phases  of  its  life  history. 

(d)  That  from  food,  as  also  from  contained  organic  mat- 
ter of  particular  soils,  such  micro-organism  can  manufacture, 
by  the  chemical  changes  wrought  therein  through  certain 
of  its  life  processes,  a  substance  which  is  a  virulent  chemical 
poison. 

Here,  then,  we  have  a  large  mass  of  evidence  from  the 
data  collected  by  Buchanan,  Bowditch,  Pettenkofer,  and 
Ballard.  But  much  of  this  work  was  done  anterior  to  the 
time  of  the  application  of  bacteriology  to  soil  constitution. 
Recently  the  matter  has  received  increased  attention  from 
various  workers  abroad,  and  in  England  from  Dr.  Sid- 
ney Martin,  Professor  Hunter  Stewart,  Dr.  Robertson,  and 
others.  The  greater  part  of  this  work  we  cannot  here  con- 
sider. But  some  reference  must  be  made  to  Dr.  Robertson's 
admirable  researches  into  the  growth  of  the  bacillus  of 
typhoid  in  soil.  By  experimental  inoculation  of  soil  with 
broth  cultures,  he  was  able  to  isolate  the  bacillus  twelve 
months  after,  alive  and  virulent.  He  concludes  that  the 
typhoid  organism  is  capable  of  growing  very  rapidly  in  cer- 
tain soils,  and  under  certain  circumstances  can  survive  from 
one  summer  to  another.  The  rains  of  spring  and  autumn 
or  the  frosts  and  snows  of  winter  do  not  kill  them  off  so  long 
as  there  is  sufficient  organic  pabulum.  Sunlight,  the  bac- 
tericidal power  of  which  is  well  known,  had,  as  would  be 
expected,  no  effect  except  upon  the  bacteria  directly  ex- 
posed to  its  rays.  The  bacillus  typhosus  quickly  dies  out 
in  the  soil  of  grass-covered  areas.  Dr.  Robertson  holds 
that  the  chief  channel  of  infection  between  typhoid-infected 
soil  and  man  is  dust.  As  in  tubercle  and  anthrax,  so  in 
typhoid,  dried  dust  or  excreta  containing  the  bacillus  is  the 
vehicle  of  disease. 

Hitherto  we  have  addressed  ourselves  to  those  diseases 
the  known  causal  organisms  of  which  reside,  normally  or 


BACTERIA    IN   THE   SOIL 


177 


abnormally,  in  the  soil.  But  closely  allied  to  these  matters 
connected  with  the  role  of  pathogenic  bacteria  in  soil  is  the 
question  of  what  has  been  termed  the  miasmatic  influence 
of  soil.  The  term  "  miasm  "  has  had  an  extensive  and 
somewhat  diffuse  application  in  medical  science.  It  may 
happen  in  the  future  that  typhoid  will  be  classified  strictly 
as  a  miasmatic  disease.  But  at  present,  in  the  transition 
state  of  the  science,  it  would  hardly  be  justifiable  to  classify 
typhoid  with  a  typically  miasmatic  disease  like  malaria. 
Yet  it  is  clear  that  mention  should  here  be  made  of  a  group 
of  diseases  of  which  malaria  is  the  type,  and  of  which  the 
tropics  generally  are  the  native  land.  The  bacterial  etiology 
of  the  group  is  by  no  means  worked  out.  The  cause  of 
malaria  alone  is  not  yet  a  closed  subject.  However  the  de- 
tails of  the  etiology  of  this  group  finally  arrange  themselves, 
there  is  little  doubt  of  two  facts,  viz.,  the  diseases  are  prob- 
ably produced  by  bacteria  or  allied  protozoa,  and  soil  plays 
an  important  part  in  their  production. 

From  what  has  been  said,  it  will  be  seen  that  though  a 
considerable  amount  of  knowledge  has  been  obtained  re- 
specting bacteria  in  the  soil,  it  may  be  conjectured  that 
actually  there  is  still  a  great  deal  to  ascertain  before  the 
micro-biology  of  soil  is  in  any  measure  complete  or  even 
intelligent.  The  mere  mention  of  tetanus  and  typhoid  in  the 
soil,  and  their  habits,  nutriment,  and  products  therein,  not 
to  mention  the  work  of  the  economic  bacteria,  is  to  open  up 
to  the  scientific  mind  a  vast  realm  of  possibility.  It  is 
scarcely  too  much  to  say  that  a  fuller  knowledge  of  the  part 
which  soil  plays  in  the  culture  and  propagation  of  bacteria 
may  suffice  to  revolutionise  the  practice  of  preventive  medi- 
cine. Truly,  our  knowledge  at  the  moment  is  rather  a 
heterogeneous  collection  of  isolated  facts  and  theories,  some 
of  which,  at  all  events,  require  ample  confirmation;  still, 
there  is  a  basis  for  the  future  which  promises  much  con- 
structive work. 


CHAPTER  VI 


BACTERIA  IN  MILK,  MILK  PRODUCTS,  AND 
OTHER  FOODS 

INJURIOUS  micro-organisms  in  foods  are,  fortunately  for 
I  the  consumers,  usually  killed  by  cooking.  Vast  num- 
bers are,  as  far  as  we  know,  of  no  harm  whatever.  Alarming 
reports  of  the  large  numbers  of  bacteria  which  are  contained 
in  this  or  that  food  are  generally  as  irrelevant  as  they  are 
incorrect.  Bacteria,  as  we  have  seen,  are  ubiquitous.  In 
food  we  have  abundance  of  the  chief  thing  necessary  to 
their  life  and  multiplication — favourable  nutriment.  Hence 
we  should  expect  to  find  in  uncooked  or  stale  food  an  ample 
supply  of  saprophytic  bacteria.  There  was  much  wholesome 
truth  in  the  assertions  made  some  two  years  ago  by  the  late 
Professor  Kanthack,  to  the  effect  that  good  food  as  well  as 
bad  frequently  contained  large  numbers  of  bacteria,  and 
often  of  the  same  species.  It  is  well  that  we  should  be- 
come familiarised  with  this  idea,  for  its  accuracy  cannot  be 
doubted,  and  its  usefulness  at  the  present  time  may  not 
be  without  its  beneficial  effect. 

Nevertheless,  it  is  well  we  should  know  the  bacterial  flora 
of  good  and  bad  foods  for  at  least  two  reasons.  First,  there 
is  no  doubt  whatever  that  a  considerable  number  of  cases  of 
poisoning  can  be  traced  every  year  to  food  containing  harm- 
ful bacteria  or  their  products.  To  several  of  the  more 
notorious  cases  we  shall  have  occasion  to  refer  in  passing. 
Secondly,  we  may  approach  the  study  of  the  bacteriology 
of  foods  with  some  hope  that  therein  light  will  be  found 

178 


BACTERIA    IN  FOODS  179 

upon  some  important  habits  and  effects  of  microbes.  There 
can  be  little  doubt  that  food-bacteria  afford  an  example  of 
association  and  antagonism  of  organisms  to  which  reference 
has  already  been  made.  Any  information  that  can  be 
gleaned  to  illumine  these  abstruse  questions  would  be  very 
welcome  at  the  present  time.  But  there  is  a  still  further, 
and  possibly  an  equally  important,  point  to  bear  in  mind, 
namely,  the  economic  value  of  microbes  in  food.  In  a  short 
account  like  the  present  it  will  be  impossible  to  enter  into 
hypotheses  of  pathology,  but  we  shall  at  least  be  able  to 
consider  some  of  these  interesting  experiments  which  have 
been  conducted  in  the  sphere  of  beneficial  bacteria. 

The  injurious  effects  of  organisms  contained  in  foods  has 
been  elucidated  by  the  excellent  work  of  the  late  Dr.  Bal- 
lard.  From  the  careful  study  of  a  number  of  epidemics  due 
to  food  poisoning,  this  patient  observer  was  able,  without 
the  aid  of  modern  bacteriology,  to  arrive  at  a  simple  prin- 
ciple which  must  not  be  forgotten.  'Food  poisoning  is  due 
either  to  bacteria  themselves  or  to  their  products,  which  are 
contained  in  the  substance  of  the  food.  In  cases  of  the  first 
kind,  bacteria  gaining  entrance  to  the  human  alimentary 
canal,  set  up  their  specific  changes  and  produce  their  toxins, 
and  by  so  doing  in  course  of  time  bring  about  a  diseased 
condition,  with  its  consequent  symptoms.  On  the  other 
hand,  if  the  products,  sometimes  called  ptomaines,  are  in- 
gested as  such,  the  symptoms  set  up  by  their  action  in  the 
body  tissues  appear  earlier.  From  these  facts  Dr.  Ballard 
deduced  the  simple  principle  that  if  there  is  no  incubation 
period  or,  at  all  events,  a  comparatively  short  space  of  time 
between  eating  the  poisoned  food  and  the  advent  of  disease, 
the  agents  of  the  disease  are  products  of  bacteria.  If,  on  the 
other  hand,  there  is  an  incubation  period,  the  agents  are 
probably  bacteria. 

It  is  necessary  to  mention  two  other  facts.     Dr.  Cautley  l 

1  Report  of  Medical  Officer  to  Local  Government  Board,  1895-1896,  Appendix. 


ISO  BACTERIA 

has  recently  been  engaged  in  isolating  from  poisoned  foods 
the  different  species  of  bacteria  present.  It  would  appear 
that  these  are  limited,  as  a  rule,  to  two  or  three  Icinds.  As 
regards  disease,  the  organisms  of  suppuration  are  the  most 
common.  Liquefying  or  fermentative  bacteria  are  fre- 
quently present,  the  Proteus  family  being  well  represented. 
In  addition  there  are,  according  to  circumstances,  a  number 
of  common  saprophytes.  Now,  as  we  have  pointed  out, 
these  organisms  may  act  injuriously  by  some  kind  of  co- 
operation, or  they  may  by  themselves  be  harmless,  and 
pathological  conditions  be  due  to  the  occasional  introduc- 
tion of  pathogenic  species. 

The  other  fact,  requiring  recognition  from  anyone  who 
proposes  to  study  the  bacteriology  of  foods,  is  that  a  certain 
appreciable  amount  of  the  responsibility  for  food  poisoning 
rests  with  the  tissues  of  the  individual  ingesting  the  food. 
There  is  ample  evidence  in  support  of  the  fact  that  not  all 
the  persons  partaking  of  infected  food  suffer  equally,  and 
occasionally  some  escape  altogether.  We  know  little  or 
nothing  of  the  causes  of  such  modification  in  the  effect 
produced.  It  may  be  due  to  other  organisms,  or  chemical 
substances  already  in  the  alimentary  canal  of  the  individual, 
or  it  may  be  due  to  some  insusceptibility  or  resistance  of 
the  tissues.  Be  that  as  it  may,  it  is  a  matter  which  must 
not  be  neglected  in  estimating  the  effects  of  food  contamin- 
ated with  bacteria  or  their  products. 

Milk.  There  are  few  liquids  in  general  use  which  contain 
such  enormous  numbers  of  germs  as  milk.  To  begin  with, 
milk  is  in  every  physical  way  admirably  adapted  to  be  a 
favourable  medium  for  bacteria.  It  is  constituted  of  all  the 
chief  elements  of  the  food  upon  which  bacteria  live.  It  is 
frequently  at  a  temperature  favourable  to  their  growth.  It 
is  par  excellence  an  absorptive  fluid.  A  dish  of  ordinary 
water  and  a  dish  of  newly  drawn  milk  laid  side  by  side,  and 
under  similar  conditions  of  temperature,  will  rapidly  demon- 


BACTERIA    IN  FOODS  l8l 

strate  the  difference  in  degree  of  absorptivity  between  the 
two  fluids.  Yet,  whilst  this  general  fact  is  true,  we  must 
emphasise  at  the  outset  the  possibility  and  practicability  of 
securing  absolutely  pure  sterile  milk.  Recently  some  milk- 
ing was  carried  out  under  strict  antiseptic  precautions,  with 
the  above  sterile  result.  The  udder  was  thoroughly  cleansed, 
the  hands  of  the  milker  washed  with  corrosive  sublimate  and 
then  pure  water,  the  vessels  which  were  to  receive  the  milk 
had  been  carefully  sterilised,  and  the  whole  process  was 
carried  out  in  strict  cleanliness.  The  result  was  that  the 
sample  of  milk  remained  sweet  and  good  and  contained  no 
germs.  It  should  be  stated  that  the  first  flow  of  milk,  wash- 
ing out  the  milk-ducts  of  the  udder,  was  rejected.  This 
fact  of  the  sterility  of  cleanly  drawn  milk  is  not  a  new  one, 
and  has  been  established  by  many  bacteriologists.  Milk, 
then,  is  normally  a  sterile  secretion.  How  does  it  gain  its 
enormously  rich  flora  of  bacteria  ? 

Sources  of  Pollution  of  Milk.  These  are  various,  and  de- 
pend upon  many  minor  circumstances  and  conditions.  For 
all  practical  purposes  there  are  three  chief  opportunities  be- 
tween the  cow  and  the  consumer  when  milk  may  become 
contaminated  with  bacteria: 

1.  At  the  time  of  milking. 

2.  During  transit  to  the  town,  or  dairy,  or  consumer. 

3.  After  its  arrival. 

Pollution  at  the  Time  of  Milking  arises  from  the  animal, 
the  milker,  or  unclean  methods  of  milking.  It  is  now  well 
known  that  in  tuberculosis  of  the  cow  affecting  the  udder 
the  milk  itself  shows  the  presence  of  the  bacillus  of  tubercle. 
In  a  precisely  similar  manner  all  bacterial  diseases  of  the 
cow  which  affect  the  milk-secreting  apparatus  must  inevit- 
ably add  their  quota  of  bacteria  to  the  milk.  To  this  mat- 
ter we  shall  have  occasion  to  refer  again.  There  is  a  further 
contamination  from  the  animal  when  it  is  kept  unclean,  for 
it  happens  that  the  unclean  coat  of  a  cow  will  more  materi- 


1 82  BACTERIA 

ally  influence  the  number  of  micro-organisms  in  the  milk 
than  the  popularly  supposed  fermenting  food  which  the 
animal  may  eat.  It  is  from  this  external  source  rather  than 
from  the  diet  that  organisms  occur  in  the  milk.  The  hairy 
coat  offers  many  facilities  for  harbouring  dust  and  dirt.  The 
mud  and  filth  of  every  kind  that  may  be  habitually  seen  on 
the  hinder  quarters  of  cattle  all  contribute  largely  to  pol- 
luted milk.  Nor  is  this  surprising.  Such  filth  at  or  near 
the  temperature  of  the  blood  is  an  almost  perfect  environ- 
ment for  many  of  the  putrefactive  bacteria. 

The  milker  is  also  a  source  of  risk.  His  hands,  as  well  as 
the  clothes  he  is  wearing,  can  and  do  readily  convey  both 
innocent  and  pathogenic  germs  to  the  milk.  Clothed  in 
dust-laden  garments,  and  frequently  characterised  by  dirty 
hands,  the  milker  may  easily  act  as  an  excellent  purveyor 
of  germs.  Not  a  few  cases  are  also  on  record  where  it  ap~ 
pears  that  milkers  have  conveyed  germs  of  disease  from 
some  case  of  infectious  disease,  such  as  scarlet  fever,  in 
their  homes.  But  under  the  more  efficient  registration  of 
such  disease  which  has  recently  characterised  many  dairy 
companies,  the  danger  of  infection  from  this  source  has  been 
reduced  to  a  minimum.  The  habit  of  moistening  the  hands 
with  a  few  drops  of  milk  previous  to  milking  is  one  to  be 
strongly  deprecated. 

Professor  Russell  recounts  a  simple  experiment  which 
clearly  demonstrates  these  simple  but  effective  sources  of 
pollution : 

"  A  cow  that  had  been  pastured  in  a  meadow  was  taken  for 
the  experiment,  and  the  milking  done  out  of  doors,  to  eliminate 
as  much  as  possible  the  influence  of  germs  in  the  barn  air. 
Without  any  special  precaution  being  taken  the  cow  was  partially 
milked,  and  during  the  operation  a  covered  glass  dish,  contain- 
ing a  thin  layer  of  sterile  gelatine,  was  exposed  for  sixty  seconds 
underneath  the  belly  of  the  cow  in  close  proximity  to  the  milk- 
pail.  The  udder,  flank,  and  legs  of  the  cow  were  then  thor- 


BACTERIA    IN  FOODS  183 

oughly  cleaned  with  water,  and  all  of  the  precautions  referred  to 
before  were  carried  out,  and  the  milking  then  resumed.  A 
second  plate  was  then  exposed  in  the  same  place  for  an  equal 
length  of  time,  a  control  also  being  exposed  at  the  same  time  at 
a  distance  of  ten  feet  from  the  animal  and  six  feet  from  the 
ground  to  ascertain  the  germ  contents  of  the  surrounding  air. 
From  this  experiment  the  following  instructive  data  were  gath- 
ered. Where  the  animal  was  milked  without  any  special  precau- 
tions being  taken  there  were  3250  bacterial  germs  per  minute 
deposited  on  an  area  equal  to  the  exposed  top  of  a  ten-inch  milk- 
pail.  Where  the  cow  received  the  precautionary  treatment  as 
suggested  above,  there  were  only  115  germs  per  minute  deposited 
on  the  same  area.  In  the  plate  that  was  exposed  to  the  surround- 
ing air  at  some  distance  from  the  cow  there  were  65  bacteria. 
This  indicates  that  a  large  number  of  organisms  from  the  dry 
coat  of  the  animal  can  be  kept  out  of  milk  if  such  simple  pre- 
cautions as  these  are  carried  out."  l 

The  influence  of  the  barn  air,  and  the  cleanliness  or  other- 
wise of  the  barn,  is  obviously  great  in  this  matter.  As  we 
have  seen,  moist  surfaces  retain  any  bacteria  lodged  upon 
them ;  but  in  a  dry  barn,  where  molecular  disturbance  is  the 
rule  rather  than  the  exception,  it  is  not  surprising  that  the 
air  is  heavily  laden  with  microbic  life.  Here  again  many 
improvements  have  been  made  by  sanitary  cleanliness  in 
various  well-known  dairies.  Still  there  is  much  more  to  be 
done  in  this  direction  to  ensure  that  the  drawn  milk  is  not 
polluted  by  a  microbe-impregnated  atmosphere. 

The  risks  in  transit  differ  according  to  many  circumstances. 
Probably  the  commonest  source  of  contamination  is  in  the 
use  of  unclean  utensils  and  milk-cans.  Any  unnecessary 
delay  in  transit  affords  increased  opportunity  for  multipli- 
cation ;  particularly  is  this  the  case  in  the  summer  months, 
for  at  such  times  all  the  conditions  are  favourable  to  an 
enormous  increase  of  any  extraneous  germs  which  may  have 

1  H.  L.  Russell,  Dairy  Bacteriology,  p.  46. 


1 84  BACTERIA 

gained  admittance  at  the  time  of  milking.  Thus  we  have 
(i)  the  milk  itself  affording  an  excellent  medium  and  supply- 
ing ideal  pabulum  for  bacteria,  (2)  a  more  or  less  lengthened 
railway  journey  or  period  of  transit  giving  ample  time  for 
multiplication,  (3)  the  favourable  temperature  of  summer 
heat.  We  shall  refer  again  to  the  rate  of  multiplication  of 
germs  in  milk. 

Lastly,  many  are  the  advantages  given  to  bacteria  when 
milk  has  reached  its  commercial  destination.  In  milk-shops 
and  in  the  home  there  are  not  a  few  risks  to  be  added  on  to 
the  already  imposing  category.  Water  is  occasionally,  if 
not  frequently,  added  to  milk  to  increase  its  volume.  Such 
water  of  itself  will  make  its  own  contribution  to  the  flora  of 
the  milk,  unless  indeed,  which  is  unlikely,  the  water  has 
been  recently  and  thoroughly  boiled  before  addition  to 
the  milk.  Again,  it  is  impossible  to  suppose  that  in 
small  homes — perhaps  of  only  one  room — where  the  milk 
stands  for  several  hours,  pollution  is  avoidable.  From  a 
hundred  different  sources  such  milk  runs  the  risk  of  being 
polluted. 

Before  proceeding,  a  word  must  be  said  respecting  the 
first  milk  which  flows  from  the  udder  in  the  process  of  milk- 
ing, and  which  is  known  as  the  fore-milk.  This  portion  of 
the  milk  is  always  rich  in  bacterial  life  on  account  of  the 
fact  that  it  has  remained  in  the  milk-ducts  since  the  last 
milking.  However  thorough  the  manipulation,  there  will 
always  be  a  residue  remaining  in  the  ducts,  which  will,  and 
does,  afford  a  suitable  nidus  and  incubator  for  organisms. 
The  latter  obtain  their  entrance  through  the  imperfectly 
closed  teat  of  the  udder,  and  pass  readily  into  the  milk-duct, 
sometimes  even  reaching  the  udder  itself  and  setting  up 
inflammation  (mastitis).  Professor  Russell  states  that  he 
has  found  2800  germs  in  the  fore-milk  in  a  sample  of  which 
the  average  was  only  330  per  cc.  Schultz  found  83,000 
micro-organisms  per  cc.  in  the  fore-milk,  and  only  9000  in 


BACTERIA    IN  FOODS  185 

the  mid-milk.  As  a  matter  of  fact,  most  of  this  large  num- 
ber belong  to  the  lactic-acid  fermentation  group,  and  the 
fore-milk  rarely  contains  more  than  two  or  three  species,  and 
still  more  rarely  any  disease-producing  bacteria.  Still,  they 
occur  in  such  enormous  numbers  that  their  addition  to  the 
ordinary  milk  very  materially  alters  its  quality.  Bolley  and 
Hall,  of  North  Dakota,  report  sixteen  species  of  bacteria  in 
the  fore-milk,  twelve  of  which  produced  an  acid  reaction. 
Dr.  Veranus  Moore,  of  the  United  States  Department  of 
Agriculture,1  concludes  from  a  large  mass  of  data  that 
freshly  drawn  fore-milk  contains  a  variable  but  generally 
enormous  number  of  bacteria,  but  only  several  species,  the 
last  milk  containing,  as  compared  with  the  fore-milk,  very 
few  micro-organisms.  The  bacteria  which  become  localised 
in  the  milk-ducts,  and  which  are  necessarily  carried  into  the 
milk,  are  for  the  greater  part  rapidly  acid-producing  organ- 
isms, i.  e.,  they  ferment  milk-sugar,  forming  acids.  They 
do  not  produce  gas.  Still  their  presence  renders  it  neces- 
sary to  "  pasteurise  "  as  soon  as  possible.  Dr.  Moore  holds 
that  much  of  the  intestinal  trouble  occurring  in  infants  fed 
with  ordinarily  "  pasteurised  "  milk  arises  from  acids  pro- 
duced by  these  bacteria  between  the  drawing  of  the  milk 
and  the  pasteurisation. 

The  Number  of  Bacteria  in  Milk.  From  all  that  has  been 
said  respecting  the  sources  of  pollution  and  the  favourable 
nidus  which  milk  affords  for  bacteria,  it  is  not  surprising 
that  a  very  large  number  of  germs  are  almost  always  present 
in  milk.  The  quantitative  estimation  of  milk  appears  more 
alarming  than  the  qualitative.  It  is  true  some  diseases  are 
conveyed  by  bacteria  in  milk,  but  on  the  whole  most  of  the 
species  are  non-pathogenic.  Nor  need  the  numbers,  though 
serious,  too  greatly  alarm  us,  for,  as  we  shall  see  at  a  later 
stage,  disease  is  a  complicated  condition,  and  due  to  other 
agencies  and  conditons  than  merely  the  bacteria,  which  may 

1  Bureau  of  Animal  Industry  Reports,  1895-1896. 


1 86  BACTERIA 

be  the  vera  causa.  In  addition  to  the  fact  that  the  high 
numbers  have  but  a  limited  significance,  we  must  also  re- 
member that  there  is  no  uniformity  whatever  in  these  num- 
bers. The  conditions  which  chiefly  control  them  are  (i) 
temperature,  (2)  time. 

The  Influence  of  Temperature.  We  have  already  noticed, 
when  considering  the  general  conditions  affecting  bacteria, 
how  potent  an  agent  in  their  growth  is  the  surrounding 
temperature.  Generally  speaking,  temperature  at  or  about 
blood-heat  favours  bacterial  growth.  Freudenreich  has 
drawn  up  the  following  table  which  graphically  sets  forth 
the  effect  of  temperature  upon  bacteria  in  milk : 

3  hours.     6  hours.      9  hours.  24  hours. 

59°  F i  +    ...        2.5  ...          5  ...  163 

77°  F 2         ...      18.5  ...       107  ...  62,100 

95°  F 4        •••    1,290  ...   3,800  ...  5,370 

This  instructive  table  claims  some  observations.  It  will 
be  noticed  that  at  59°  F.  there  is  very  little  multiplication. 
That  may  be  accepted  as  a  rule.  At  77°  F.  the  multiplica- 
tion, though  not  particularly  rapid  at  the  outset,  results 
finally,  at  the  end  of  the  twenty-four  hours,  in  the  maxi- 
mum quantity.  These  were  probably  common  species  of 
saprophytic  bacteria,  which  increase  readily  at  a  comparat- 
ively low  temperature.  During  the  subsequent  hours,  after 
the  twenty-four,  we  should  expect  a  decline  rather  than  an 
increase  in  62,000,  owing  to  the  keen  competition  consequent 
upon  the  limitation  of  the  pabulum.  From  a  consideration 
of  these  figures  we  conclude  that  a  warm  temperature,  some- 
what below  blood-heat,  is  most  favourable  to  multiplication 
of  bacteria  in  milk ;  that  the  common  saprophytic  organisms 
multiply  the  most  rapidly;  that,  in  the  course  of  time/ 
competition  kills  off  a  large  number. 

Let  us  take  another  example,  from  Professor  Conn : 


BACTERIA    IN  FOODS  l8/ 

77°  F.  95°  F. 
2  hours  after  milking ....     (liquefied  the 

plate  of  gelatine) 1,275,000 

6             •  . . . .    14,620,000  ....  45,900,000 

9              36,55°>000  57,800,000 

24  13,702,000,000  13,812,500,000 

[Bacteria  per  cub.  inch.] 

These  almost  incredibly  large  figures  illustrate  much  the 
same  points,  particularly  the  rapid  multiplication  at  blood- 
heat,  and  the  later  rise  at  77°  C. 

The  Influence  of  Time  is  not  less  marked  than  that  of 
temperature,  as  the  following  table  will  show : 

Milk  drawn  at  59°  C.  =  153,000  m.o.  per  cub.  in. 

After  i  hour  =  616,000     "      " 

2  hours  =  539,000     "      "       " 

"      4     "  =  680,000     "      "       "      " 

"     7     "  =  1,020,000     "      "       "      " 

9  =  2,040,000 

"    24     "  =  85,000,000     "      "       "      " 

(Conn.) 

Freudenreich  gives  another  example,  as  follows : 


Milk  drawn  at  15.5°  C.  =  27,000  m.o.  per  cc. 

After    4  hours  =  34,ooo     "      "      " 

9  =  100,000     "      "      " 

"      24     "  =  4,000,000     "      "      " 

Concerning  these  figures  little  comment  is  necessary. 
But  here  again,  we  may  remember  that  this  rapid  multipli- 
cation continues  only  up  to  a  certain  point,  after  which 
competition  brings  about  a  marked  reduction. 

The  effect  of  temperature  and  time  has  been  illustrated 
by  Dr.  Buchanan  Young's  recent  researches,  laid  before  the 


1 88  BACTERIA 

Royal  Society  of  Edinburgh.  He  estimated  that  in  the 
Edinburgh  milk  supply  three  hours  after  milking  there 
were  24,000  micro-organisms  per  cc.  in  winter ;  44,000  in 
spring ;  173,000  in  late  summer  and  autumn.  Again,  he 
found  that  five  hours  after  milking  there  were  41,000  micro- 
organisms per  cc.  in  country  milk,  and  more  than  350,000 
micro-organisms  per  cc.  in  town  milk.  Many  London  milks 
would  exceed  500,000  per  cc.1 

There  is  no  standard  or  uniformity  in  the  numerical  esti- 
mation of  bacteria  in  milk.  A  host  of  observers  have  re- 
corded widely  varying  returns  due  to  the  widely  varying 
circumstances  under  which  the  milk  has  been  collected, 
removed,  stored,  and  examined.  Nor  is  it  possible  to  es- 
tablish any  standard  which  may  be  accepted  as  a  normal 
or  healthy  number  of  bacteria,  as  is  done  in  water  examin- 
ation. Bitter  has  suggested  50,000  micro-organisms  per  cc. 
as  a  maximum  limit  for  milk  intended  for  human  consump- 
tion. 

But  owing  to  differences  of  nomenclature  and  classification, 
in  addition  to  differences  in  mode  of  examination  at  present 
existing  in  various  countries,  it  is  impossible  to  state  even 
approximately  how  many  bacteria  and  how  many  species  of 
bacteria  have  been  isolated  from  milk.  Until  some  com- 
mon international  standard  is  established  mathematical 
computations  are  practically  worthless.  They  are  needlessly 
alarming  and  sensational.  And  it  should  be  remembered 
that  great  reliance  cannot  be  placed  upon  these  numerical 
estimations.  They  vary  from  day  to  day,  and  even  hour  to 
hour.  Furthermore,  vast  numbers  of  bacteria  are  economic 
in  the  best  sense  of  the  term,  and  the  bacteria  of  milk  are 
chiefly  those  of  a  fermentative  kind,  and  not  disease- 
producers. 

Kinds  of  Bacteria  in  Milk.  It  is  clear  from  the  foregoing 
that  the  only  valuable  estimation  of  bacteria  in  milk  is  a 

1  British  Medical  Journal,  1895,  vol.  ii.,  p.  322. 


BACTERIA   IN  FOODS  189 

qualitative  one.     The  kinds  commonly  found  may  be  class- 
ified thus: 

1.  Non-pathogenic;  fermenting  and  various  unclassified 
micro-organisms. 

2.  Pathogenic;    tuberculosis,    typhoid,    cholera,    scarlet 
fever,   diphtheria,   and  suppurative  diseases  have  all  been 
spread  by  the  agency  of  milk. 

i.    The  Fermentation  Bacteria 

At  the  most  we  can  make  a  merely  provisional  classifica- 
tion of  these  processes.  Many  of  them  are  intimately  re- 
lated. Of  others,  again,  our  knowledge  is  at  present  very 
limited.  It  may  be  advisable,  before  proceeding,  to  con- 
sider shortly  what  are  the  constituents  of  milk  upon  which 
living  ferments  of  various  kinds  exert  their  action.  A 
tabulation  of  the  chief  constituents  would  be  as  follows: 

f  (i)  Water 87.5  per  cent. 

Ordinary  (2)  Milk-sugar 4.9    "       " 

fresh  milk  =    <j    (3)  Fat 3.6    "       " 

100  per  cent.        (4)  Proteids  (casein,  etc.). .  3.3    "       " 

[  (5)  Mineral  matter 0.7    "       " 

100.0 

Another  mode  of  expressing  average  milk  constitution 
would  be  thus: 

Fat 4.1  per  cent. 

Solids  not  fat..  8.8    "       " 


12.9    " 


It  is  probably  too  obvious  to  need  remark  that  milks  vary 
in  standard,  but  the  above  figures  may  be  taken  as  authentic 
averages. 


I QO  BACTERIA 

Milk-sugar,  or  Lactose  (C,3H34O12).  This  is  an  import- 
ant and  constant  constituent  of  milk.  It  forms  the  chief 
substance  in  solution  in  whey  or  serum.  Milk-sugar  ap- 
proximates to  dextrose  in  its  action  on  polarised  light.  By 
boiling  with  sulphuric  acid  it  is  converted  into  dextrose  and 
galactose. 

Fat  occurs  in  milk  as  suspended  globules,  and  by  churning 
may  be  made  into  butter. 

The  Proteids  include  casein,  albumen,  lactoprotein,  and  a 
small  quantity  of  globulin.  These  are  the  nitrogenous 
bodies. 

Mineral  Matter.  The  ash  of  milk,  obtained  by  careful 
ignition  of  the  solids,  contains  calcium,  magnesium,  potas- 
sium, sodium,  phosphoric  acid,  sulphuric  acid,  chlorine,  and 
iron,  phosphoric  acid  and  lime  being  present  in  the  largest 
amounts. 

(i)  Lactic  Acid  Fermentation.  If  milk  is  left  undisturbed, 
it  is  well  known  that  eventually  it  becomes  sour.  The  casein 
is  coagulated,  and  falls  to  the  bottom  of  the  vessel;  the 
whey  or  serum  rises  to  the  top.  In  fact,  a  coagulation 
analogous  to  the  clotting  of  blood  has  taken  place.  In 
addition  to  this,  the  whole  has  acquired  an  acid  taste.  Now, 
this  double  change  is  not  due  to  any  one  of  the  constituents 
we  have  named  above.  It  is,  in  short,  a  fermentation  set 
up  by  a  living  ferment  introduced  from  without.  The  con- 
stituent most  affected  by  the  ferment  is  the  milk-sugar, 
which  is  broken  down  into  lactic  acid,  carbonic  acid  gas, 
and  other  products. 

For  many  years  it  has  been  known  that  sour  milk  con- 
tained bacteria.  Pasteur  first  described  the  Bacillus  acidi 
lactici,  which  Lister  isolated  and  obtained  in  pure  culture. 
Hueppe  contributed  still  further  to  what  was  known  of  this 
bacillus,  and  pointed  out  that  there  were  a  large  number  of 
varieties,  rather  than  one  species,  to  be  included  under  the 
term  B.  acidi  lactici.  We  have  already  seen  that  these 


BACTERIA   IN  FOODS  191 

bacilli  do  not  as  a  rule  liquefy  gelatine,  form  spores,  are 
non-motile,  and  are  easily  killed  by  heat. 

When  a  certain  quantity  of  lactic  acid  has  been  formed 
the  fermentation  ceases.  It  will  recommence  if  the  liquid 
be  neutralised  with  carbonate  of  lime,  or  pepsine  added. 
Since  Pasteur's  discovery  of  a  causal  bacillus  for  this  ferment- 
ation, other  investigators  have  added  a  number  of  bacteria 
to  the  lactic  acid  family.  Some  of  these  in  pure  culture 
have  been  used  in  dairy  industry  to  add  to  the  butter  a  pure 
sour  taste,  a  more  or  less  aromatic  odour,  and  a  higher 
degree  of  preservation. 

(2)  Butyric  Acid  Fermentation.      This  form  of  ferment- 
ation is  also  one  which  we  have  previously  considered. 

Both  in  lactic  and  butyric  fermentation  we  must  recognise 
that  in  the  decomposition  of  milk-sugar  there  are  almost 
always  a  number  of  minor  products  occurring.  Some  of  the 
chief  of  these  are  gases.  Hydrogen,  carbonic  acid,  nitrogen, 
and  methane  occur,  and  cause  a  characteristic  effect  which 
is  frequently  deleterious  to  the  flavour  of  the  milk  and  its 
products.  Most  of  the  gas-producing  ferments  are  members 
of  the  lactic  acid  group,  and  are  sometimes  classified  in  a 
group  by  themselves.  In  cheese-making  the  gases  create 
the  pin-holes  and  air-spaces  occasionally  seen. 

(3)  Curdling  Fermentations  without  Acid  Production.     Of 
these  there  are  several,  caused  by  different  bacteria.     What 
happens  is  that  the  milk  coagulates,  as  we  have  described, 
but  no  acid  is  produced,  the  whey  being  sweet  to  the  taste 
rather  than  otherwise.     Digestion  of  casein  may  or  may  not 
take  place. 

We  must  now  mention  several  fermentations  about  which 
little  is  known.  They  are  designated  by  terms  denoting 
the  outward  condition  of  the  milk,  without  giving  any  in- 
formation respecting  the  real  physiological  alteration  which 
has  occurred. 

(4)  Bitter  Fermentation.     Some  bitter  conditions  of  milk 


192  BACTERIA 

are  due  to  irregularity  of  diet  in  the  cow.  Similar  changes 
occur  in  conjunction  with  some  of  the  acid  fermentations. 
Weigmann  and  Conn  have,  however,  shown  that  there  is  a 
specific  bitterness  in  milk  due  to  bacteria  which  appear  to 
produce  no  other  change.  Hueppe  suggests  that  it  may 
be  due  in  part  to  a  proteid  decomposition  resulting  in  bitter 
peptones.  There  seems  to  be  some  evidence  for  supposing 
that  the  bitter  bacteria  produce  very  resistant  spores,  which 
enable  them  to  withstand  treatment  under  which  the  lactic 
acid  succumbs. 

(5)  Slimy  Fermentation.  This  graphic  but  inelegant  word 
is  used  to  denote  an  increased  viscosity  in  milk,  and  its 
tendency  when  being  poured  to  become  ropy  and  fall  in 
strings.  Such  a  condition  deprives  the  milk  of  its  use  in  the 
making  of  certain  cheeses,  whilst  in  other  cases  it  favours 
the  process.  In  Holland,  for  example,  in  the  manufacture 
of  Edam  cheese,  this  "  slimy  "  fermentation  is  desired. 
Tcettemcelk,  a  popular  beverage  in  Norway,  is  made  from 
milk  that  has  been  infected  with  the  leaves  of  the  common 
butter  wort,  Pinguicula  vulgaris,  from  which  Weigmann 
separated  a  bacillus  possessing  the  power  of  setting  up  slimy 
fermentation.  There  are,  perhaps,  as  many  as  a  dozen 
species  of  bacteria  which  have  in  a  greater  or  less  degree  the 
power  of  setting  up  this  kind  of  fermentation.  In  1882 
Schmidt  isolated  the  Micrococcus  viscosus,  which  occurs  in 
chains  and  rosaries,  affecting  the  milk-sugar.  It  grows  at 
blood-heat,  and  is  not  easily  destroyed  by  cold.  Its  effect 
on  various  sugars  is  the  same.  The  M.  Freudenreichii,  the 
specific  micro-organism  of  "  ropiness  "  in  milk,  is  a  large, 
non-motile,  liquefying  coccus,  which  can  produce  its  result 
in  milk  within  five  hours.  On  account  of  its  resistance  to 
drying,  it  is  difficult  to  eradicate  when  once  it  makes  its 
appearance  in  a  dairy.  The  organism  used  in  making  Edam 
cheese  is  the  Streptococcus  Hollandicus,  and  in  hot  milk  it 
can  produce  ropiness  in  one  day.  A  number  of  bacilli  have 


BACTERIA    IN  FOODS 


193 


been  detected  by  several  observers  and  classified  as  slime 
fermentation  bacteria.  The  Bacillus  lactis  pituitosi,  a 
slightly  curved,  non-liquefying  rod,  which  is  said  to  produce 
a  characteristic  odour,  in  addition  to  causing  ropiness,  brings 
about  some  acidity.  B.  lactis  viscosus  is  slow  in  starting  its 
fermentation,  but  maintains  its  action  for  as  long  as  a 
month.  Many  of  the  above  organisms,  with  others,  pro- 
duce "  slimy  "  fermentation  in  alcoholic  beverages  as  well 
as  in  milk. 

(6)  Soapy  Milk.     This  is  still  another  form  of  fermenta- 
tion, the  etiology  of  which  has  been  elucidated  by  Weig- 
mann.     The  Bacillus  saponacei  imparts  to  milk  a  peculiar 
soapy  flavour.     It  was  detected  in  the  straw  of  the  bedding 
and  hay  of  the  fodder,  and  from  such  sources  may  infect 
the  milk.     There  is  little  or  no  coagulation. 

(7)  Chromogenic  Changes.      We   have    already  remarked 
that  colour  is  the  natural  and  apparently  only  product  of 
many  of  the  innocent  bacteria.     They  put  out  their  strength, 
so  to  speak,  in  the  production  of  bright  colours.     The  chief 
colours  produced  by  germs  in  milk  are  as  follows : 

Red  Milk.  Bacillus  prodigiosus,  in  the  presence  of  oxy- 
gen, causes  a  redness,  particularly  on  the  surface  of  milk. 
It  was  the  work  of  this  bacillus  that  caused  "  the  bleeding 
host,"  which  was  one  of  the  superstitions  of  the  Middle 
Ages.  B.  lactis  erythrogenes  produces  a  red  colour  only  in 
the  dark,  and  in  milk  that  is  not  strongly  acid  in  reaction. 
When  grown  in  the  light  this  organism  produces  a  yellow 
colour.  There  is  a  red  sarcina  (Sarcina  rosed]  which  also 
has  the  faculty  of  producing  red  pigment.  One  of  the 
yeasts  is  another  example. 

It  must  not  be  forgotten  that  redness  in  milk  may  actually 
be  due  to  the  presence  of  blood  from  the  udder  of  the  cow. 
In  such  a  case  the  blood  and  milk  will  be  inextricably  mixed 
together,  and  not  in  patches  or  a  pellicle. 

Blue  Milk  is  due  to  the  growth  of  Bacillus  cyanogenus. 


I94 


BA  CTERIA 


This  is  an  actively  motile  rod,  the  presence  of  which  does 
not  materially  affect  the  milk,  but  causes  the  milk  products 
to  be  of  poor  quality. 

Yellow  Milk.  Bacillus  synxanthus  is  held  responsible  for 
curdling  the  milk,  and  then  at  a  later  stage,  in  redissolving 
the  curd,  produces  a  yellow  pigment. 

Violet  and  Green  Pigments  in  milk  are  also  the  work  of 
various  bacteria. 

2.    Various  Unclassified  Bacteria 

% 

In  milk  this  is  a  comparatively  small  group,  for  it  happens 
that  those  bacteria  in  milk  which  cannot  be  classified  as 
fermentative  or  pathogenic  are  few.  The  almost  ubiquitous 
Bacillus  coli  communis  occurs  here  as  elsewhere,  and  might 
be  grouped  with  the  gaseous  fermentative  organisms  on  ac- 
count of  its  extraordinary  power  of  producing  gas  and  break- 
ing up  the  medium  (whether  agar  or  cheese)  in  which  it  is 
growing.  What  its  exact  role  is  in  milk  it  would  be  diffi- 
cult to  say.  It  may  act,  as  it  frequently  does  elsewhere,  by 
association  in  various  fermentations.  Some  authorities  hold 
that  its  presence  in  excessive  numbers  may  cause  epidemic 
diarrhoea  in  infants. 

Several  years  ago  a  commission  was  appointed  by  the 
British  Medical  Journal  to  inquire  into  the  quality  of  the 
milk  sold  in  some  of  the  poorer  districts  of  London.  Every 
sample  was  found  to  contain  Bacillus  coli,  and  it  was  de- 
clared that  this  particular  microbe  constituted  90  per  cent, 
of  all  the  organisms  found  in  the  milk.1  We  record  this 
statement,  but  accept  it  with  some  misgiving.  The  diag- 
nosis of  B.  coli  four  or  five  years  ago  was  not  such  a  strict 
matter  as  to-day.  Still,  undoubtedly,  this  particular  or- 
ganism is  not  uncommonly  found  in  milk,  and  its  source 
is  unclean  dairying.  In  the  same  investigation  Proteus 

1  British  Medical  Journal,  1895,  vol.  ii.,  p.  322. 


BACTERIA    IN  FOODS  195 

vulgaris,  B.  fluorescent,  and  many  liquefying  bacteria  were 
frequently  found.  Their  presence  in  milk  means  contam- 
ination with  putrefying  matter,  surface  water,  or  a  foul 
atmosphere. 

A  number  of  water  bacteria  find  their  way  into  milk  in 
the  practice  of  adulteration,  and  foul  byres  afford  ample 
opportunity  for  aerial  pollution. 

Another  unclassified  group  occasionally  present  in  milk  is 
represented  by  moulds,  particularly  Oidium  lactis,  the  mould 
which  causes  a  white  fur,  possessing  a  sour  odour.  It  is 
allied  to  the  Mycoderma  albicans  (O.  albicans),  which  also 
occurs  in  milk,  and  causes  the  whitish-grey  patches  on  the 
mucous  membrane  of  the  mouths  of  infants  (thrush}.  These 
and  many  more  are  occasionally  present  in  milk. 

3.    The  Disease-Producing  Power  of  Milk 

The  general  use  of  milk  as  an  article  of  diet,  especially  by 
the  younger  and  least  resistant  portion  of  mankind,  very 
much  increases  the  importance  of  the  question  as  to  how 
far  it  acts  as  a  vehicle  of  disease.  Recently  considerable 
attention  has  been  drawn  to  the  matter,  though  it  is  now  a 
number  of  years  since  milk  was  proved  to  be  a  channel  for 
the  conveyance  of  infectious  diseases.  During  the  last 
twenty  years  particular  and  conclusive  evidence  has  been 
deduced  to  show  that  milch  cows  may  themselves  afford  a 
large  measure  of  infection.  The  recent  extensive  work  in 
tuberculosis  by  the  Royal  Commission  has  done  much  to 
obtain  new  light  on  the  conveyance  of  that  disease  by  milk 
and  meat.  The  enormous  strides  in  the  knowledge  of 
diphtheria  and  other  germ  diseases  have  also  placed  us  in 
a  better  position  respecting  their  conveyance  by  milk. 
Generally  speaking,  for  reasons  already  given,  milk  affords 
an  ideal  medium  for  bacteria,  and  its  adaptibility  therefore 
for  conveying  disease  is  undoubted.  We  may  now  suitably 


196  BACTERIA 

turn  to  speak  shortly  of  the  outstanding  facts  of  the  chief 
diseases  carried  by  milk. 

Tuberculosis.  It  is  well  known  that  this  disease  is  not  a 
rare  one  amongst  cattle.  The  problem  of  infective  milk  is, 
however,  simplified  at  the  outset  by  recognising  the  now 
well-established  fact  that  the  milk  of  tuberculous  cows  is 
only  certainly  able  to  produce  tuberculosis  in  the  consumers 
when  the  tuberculous  disease  affects  the  udder.  This  is  not 
necessarily  a  condition  of  advanced  tuberculosis.  The  udder 
may  become  affected  at  a  comparatively  early  stage.  But 
to  make  the  milk  infective  the  udder  must  be  tubercular, 
and  milk  from  such  an  udder  possesses  a  most  extraordinary 
degree  of  virulence.  When  the  udder  itself  is  thus  the  seat 
of  disease,  not  only  the  derived  milk,  but  the  skimmed 
milk,  butter-milk,  and  even  butter,  all  contain  tuberculous 
material  actively  injurious  if  consumed.  Furthermore, 
tubercular  disease  of  the  udder  spreads  in  extent  and  degree 
with  extreme  rapidity.  From  these  facts  it  will  be  obvious 
that  it  is  of  first-rate  importance  to  be  able  to  diagnose  udder 
disease.  This  is  not  always  possible  in  the  early  stage.  The 
signs  upon  which  most  reliance  may  be  placed  are  the  en- 
largement of  the  lymph-glands  lying  above  the  posterior 
region  of  the  udder;  the  serous,  yellowish  milk  which  later 
on  discharges  small  coagula;  the  partial  or  total  lack  of 
milk  from  one  quarter  of  the  udder  (following  upon  excess- 
ive secretion);  the  hard,  diffuse  nodular  swelling  and  in- 
duration of  a  part  or  the  whole  wall  of  the  udder;  and  the 
detection  in  the  milk  of  tubercle  bacilli.  The  whole  organ 
may  increase  in  weight  as  well  as  size,  and  on  post-mortem 
examination  show  an  increase  of  connective  tissue,  a  num- 
ber of  large  nodules  of  tubercle,  and  a  scattering  of  small 
granular  bodies,  known  as  "  miliary  "  tubercles.  Tuberculin 
may  be  used  as  an  additional  test.  The  udder  is  affected 
in  about  two  per  cent,  of  tuberculous  cows. 

There  are  a  variety  of  causes  in  addition  to  the  vera  causa, 


BACTERIA    IN  FOODS  197 

the  presence  of  the  bacillus  of  tubercle,  which  make  the  dis- 
ease common  amongst  cattle.  Constitution,  temperament, 
age,  work,  food,  and  prolonged  lactation  are  the  individual 
features  which  act  as  predisposing  conditions;  they  may  act 
by  favouring  the  propagation  of  the  bacillus  or  by  weaken- 
ing the  resistance  of  the  tissues.  To  this  category  must 
further  be  added  conditions  of  environment.  Bad  stabling, 
dark,  ill-ventilated  stalls,  high  temperature,  prolonged  and 
close  contact  with  other  cows,  all  tend  in  the  same  direction. 

Though  there  can  be  no  doubt  as  to  the  virulence  of  tuber- 
culous milk,  it  may  be  remembered  with  satisfaction  that 
only  about  two  per  cent,  of  tuberculous  cows  have  unmis- 
takably tubercular  milk.  Even  of  this  tubercular  milk, 
unless  it  is  very  rich  in  bacilli  and  is  ingested  in  large  quan- 
tities, the  risks  are  practically  small  or  even  absent.  Practi- 
cally the  danger  from  drinking  raw  milk  exists  only  for 
persons  who  use  it  as  their  sole  or  principal  food,  that  is  to 
say,  young  children  and  certain  invalids.  With  adults  in 
normal  health  the  danger  is  greatly  minimised,  as  the  healthy 
digestive  tract  is  relatively  insusceptible.  Moreover,  dairy 
milk  is  almost  invariably  mixed  milk;  that  is  to  say,  if 
there  is  a  tubercular  cow  in  a  herd  yielding  tubercle  bacilli 
in  her  milk,  the  addition  of  the  milk  of  the  rest  of  the  herd 
so  effectually  dilutes  the  whole  as  to  render  it  almost 
innocuous. 

But  if  for  practical  purposes  we  look  upon  all  milk  derived 
from  tubercular  udders  as  highly  infective,  we  may  adopt  a 
comparatively  simple  and  efficient  remedy.  To  avoid  all 
danger  it  is  sufficient  to  bring  the  milk  to  a  boil  for  a  few 
minutes  before  it  is  consumed ;  in  fact,  the  temperature  of 
85°  C.  (160°  F.)  prolonged  for  five  minutes  kills  all  bacilli. 
The  common  idea  that  boiled  milk  is  indigestible,  and 
that  the  boiling  causes  it  to  lose  much  of  its  nutritive  value, 
is  largely  groundless. 

Milk  may  become  tubercular  through  the  carelessness  or 


I98 


BACTERIA 


dirty  habits  of  the  milker.  Such  a  common  practice  as 
moistening  the  hands  with  saliva  previously  to  milking  may, 
in  cases  of  tubercular  milkers,  effectually  contaminate  the 
milk.  Again,  it  may  become  polluted  by  dried  tubercular 
excreta  getting  into  it.  Such  conveyances  must  be  of  rare 
occurrence,  yet  their  possibility  should  not  be  forgotten. 

An  infant  suckled  by  a  tuberculous  mother  would  run 
similarly  serious  risks  of  becoming  infected  with  the  disease. 

In  Liverpool,  Dr.  E.  W.  Hope,  the  Medical  Officer  of 
Health,  has  organised  an  admirable  system  of  examination 
by  skilled  bacteriologists  to  find  to  what  degree  the  Liver- 
pool milk  supply  is  contaminated  with  tubercle.  The  final 
result  of  this  pioneer  work,  which  ought  really  to  be  under- 
taken by  every  great  corporation  responsible  to  the  citizens 
for  a  pure  water  and  pure  milk  supply,  is  to  the  effect  that 
in  Liverpool  5.2  per  cent,  of  the  samples  of  milk  taken 
from  the  city  shippons  contains  tubercle  bacilli.  As  regards 
the  milk  sent  in  from  the  country,  the  return  is  that  13.4 
per  cent,  is  contaminated  with  the  bacillus  of  tubercle. 


TOWN  SHIPPONS. 

COUNTRY  SHIPPONS. 

TOTAL. 

Total. 
228 

Infected. 
12 

Per  cent. 

5-2 

Total. 
67 

Infected. 

9 

Per  cent. 
13.4 

295 

Such  results  are  very  significant,  and  indicate  the  import- 
ance of  all  large  corporations  obtaining  the  service  of  system- 
atic and  periodic  bacteriological  examination  of  the  milk 
supply.  Nor  are  the  results  surprising,  for  when  we  remem- 
ber the  habits  of  the  tubercle  bacillus  we  cannot  conceive 
a  more  favourable  nurture  ground  than  the  typical  byre. 
'  Nothing  worse  than  the  insanitary  conditions  of  the  life 
of  the  average  dairy  cow,"  says  Sir  George  Brown,  late  of 
the  Board  of  Agriculture,  "  can  be  imagined."  It  will  be 
obvious  that  the  above  facts  make  it  incumbent  upon  re- 


BACTERIA    IN  FOODS  199 

sponsible  authorities  to  see  that  not  a  stone  is  left  unturned 
to  enforce  cleanliness  in  all  dairy  work,  isolation  of  dis- 
eased cows,  and  strict  treatment  of  all  infected  milk. 

Typhoid  Fever.  Jaccoud  in  France  and  Hart  in  England 
have  shown  that  enteric  fever  (typhoid)  is  not  infrequently 
spread  by  milk.  An  epidemic  affecting  386  persons  in 
Stamford,  Conn.,  U.  S.  A.,  was  traced  to  milk,  97  per  cent, 
of  the  cases  coming  from  one  single  milk  supply.  Dr. 
McNail  recently  recorded  an  outbreak  of  twenty-two  cases 
of  enteric,  due  to  a  polluted  milk  supply. 

Within  the  last  twelve  months  much  attention  has  been 
drawn  to  a  milk  source  of  typhoid  infection  by  the  epidemic 
of  typhoid  at  Bristol.  Dr.  D.  S.  Davies  has  pointed  out 
that  a  brook  received  the  sewage  of  thirty-seven  houses,  the 
overflow  of  a  cesspool  serving  twenty-two  more,  the  wash- 
ings from  fields  over  which  the  drainage  of  several  others 
was  distributed,  and  the  direct  sewage  from  at  least  one 
other,  and  then  flowed  directly  through  a  certain  farm.  The 
water  of  this  stream  supplied  the  farm  pump,  and  the  water 
itself,  it  is  scarcely  necessary  to  add,  was  highly  charged 
with  putrescent  organic  matter  and  micro-organisms.  This 
water  was  used  for  washing  the  milk-cans  from  this  particu- 
lar farm,  otherwise  the  dairy  arrangements  were  efficient. 
Part  of  the  milk  was  distributed  to  fifty-seven  houses  in 
Clifton ;  in  forty-one  of  them  cases  of  typhoid  occurred. 
Another  part  of  the  milk  was  sold  over  the  counter;  twenty 
households  so  obtaining  it  were  attacked  with  typhoid  fever, 
and  a  number  of  further  infections  and  complications  arose. 
This  evidence  would  appear  to  support  the  fact  that  milk 
may  act  in  the  same  way,  though  not  in  such  a  high  degree, 
as  water  in  the  conveyance  of  typhoid  fever. 

It  may  be  pointed  out  that  specific  typhoid  is  not  a  dis- 
ease of  animals;  consequently  no  danger  need  be  appre- 
hended from  milk  if  it  is  properly  cared  for  after  it  comes 
from  the  cow.  Typhoid  milk  is  almost  invariably  due  to 


2OO  BACTERIA 

the  addition  of  typhoid-infected  water,  either  by  way  of 
adulteration  or  in  the  process  of  washing  out  the  milk-cans. 
Cases  have,  however,  been  recorded  in  which  there  has  been 
direct  transmission  to  the  milk  from  a  person  convalescing 
from  the  disease,  and  also  indirect  transmission  by  a  milker 
serving  also  in  the  capacity  of  nurse  to  a  patient  in  his  own 
family. 

Though  the  typhoid  bacillus  appears  not  to  have  the 
power  of  multiplying  in  milk,  it  has  the  faculty  of  existing 
and  thriving  in  milk,  even  when  it  has  curdled  or  soured, 
for  a  considerable  time,  and  may  thus  infect  milk  products 
like  butter  and  cheese.  But  infection  by  milk  products  may 
be  eliminated  as  of  too  rare  occurrence  to  deserve  attention. 
The  bacillus  does  not  coagulate  the  milk  like  its  ally  the 
Bacillus  coli  communis,  which  is  a  much  more  frequent  and 
less  injurious  inhabitant  of  milk. 

Cholera.  The  cholera  bacillus,  as  we  have  already  pointed 
out,  is  unable  to  live  in  an  acid  medium.  Hence  its  life  in 
milk  is  a  limited  one,  and  generally  depends  on  some  alka- 
line change  in  the  milk.  Heim  found  that  cholera  bacilli 
would  live  in  raw  milk  from  one  to  four  days,  depending  upon 
the  temperature.  D.  D.  Cunningham,  from  the  results  of  a 
large  number  of  investigations  in  India,  concludes  that  the 
rapidly  developing  acid  fermentations  normally  or  usually 
setting  in,  connected  with  the  rapid  multiplication  of  other 
common  bacteria  and  moulds,  tend  to  arrest  the  multiplica- 
tion of  cholera  bacilli,  and  eventually  to  destroy  their  vital- 
ity. Boiling  milk  appears,  on  the  contrary,  to  increase  the 
suitability  of  milk  as  a  nidus  for  cholera  bacilli,  partly  by  its 
germicidal  effect  upon  the  acid-producing  microbes,  and 
partly  because  it  removes  from  the  milk  the  enormous  num- 
bers of  common  bacteria,  which  in  raw  milk  cause  such 
keen  competition  that  the  cholera  bacillus  finds  existence 
impossible. 

Professor  W.   J.   Simpson,  lately  the  Medical  Officer  of 


BACTERIA   IN  FOODS  2OI 

Health  for  Calcutta,  has  placed  on  record  an  interesting 
series  of  cholera  cases  on  board  the  Ardenclutha,  in  the  port 
of  Calcutta,  which  arose  from  drinking  milk  which  had  been 
polluted  with  one  quarter  of  its  volume  of  cholera-infected 
water.  This  water  came  from  a  tank  into  which  some 
cholera  dejecta  had  passed.  Of  the  ten  men  who  drank  the 
milk  four  died,  five  were  severely  ill,  and  one,  who  drank  but 
very  little  of  the  milk,  was  only  slightly  ill.  There  was  no 
illness  whatever  amongst  those  who  did  not  drink  the  milk. 

Diphtheria.  Recent  observations  on  the  infectivity  of 
diphtheria  in  milk  by  Schottelius  have  established  the  fact 
that  milk  is  a  good  medium  for  the  bacillus  of  diphtheria, 
but  that  it  rarely  acts  as  a  vehicle  for  transmitting  the  dis- 
ease. Klein  has  emphasised  the  possibility  of  this  means 
of  infection.  In  the  first  place,  it  is  obvious  that  the  milk 
may  become  infected  from  a  human  source — from  pollution 
with  diphtheritic  discharges  or  dried  ' '  fomites. ' '  Secondly, 
from  a  variety  of  different  quarters  evidence  has  been  forth- 
coming to  throw  some  suspicion  upon  the  cow  itself  as  the 
agent.  Klein  states  that  "  a  new  eruptive  disease  on  the 
teats  and  udder  of  the  cow,"  consisting  of  papules,  vesicles, 
and  induration,  may  be  set  up  by  the  subcutaneous  inocu- 
lation of  a  pure  culture  of  the  Bacillus  diphtheria.  In  these 
eruptions  a  bacillus  similar  to  the  B.  diphtheria  was  demon- 
strated. On  a  priori  grounds  this  evidence  substantiates  a 
belief  that  diphtheria,  in  some  form  or  other,  may  be  a  dis- 
ease of  cows.  Other  observers  have  not  been  able  to  confirm 
these  observations,  and  the  whole  matter  of  cow  diphtheria 
must  remain  for  the  present  sub  judice. 

As  long  ago  as  1879  W.  H.  Power  traced  an  epidemic  of 
diphtheria  in  North  London  to  the  milk  supply.  In  1887 
the  same  authority  studied  another  outbreak,  and  other 
observers  have  produced  further  evidence  in  favour  of  the 
conveyance  of  this  disease  by  milk.  Air  infection  of  milk 
by  the  Bacillus  diphtheria  probably  occurs  only  very  rarely, 


2O2  BACTERIA 

on  account  of  the  fact  that  the  organism  is  readily  killed  by 
desiccation,  and  yet  such  is  necessary  before  it  can  be  air- 
borne. The  most  frequent  mode  of  infection  of  milk  with 
this  disease  is  from  the  throats,  hands,  bodies,  or  clothing 
of  dairy  workers  suffering  from  a  mild  or  acute  form  of  the 
disease. 

The  specific  and  proved  cases  in  which  milk  has  acted  as 
the  vehicle  of  diphtheria  are,  it  is  true,  comparatively  few. 
Yet,  nevertheless,  the  possibility  of  milk  infection  in  this 
disease  is  not  one  which  we  can  afford  to  neglect. 

Scarlet  Fever.  Here  again  the  evidence  is  not  complete, 
chiefly  owing  to  the  fact  that  no  specific  organism  of  scarlet 
fever  has  yet  been  discovered.  Many  cases  have,  however, 
illustrated  the  undeniable  conveyance  of  the  disease  by 
milk.  Even  before  1881  a  number  of  milk  epidemics  of 
scarlet  fever  had  been  traced  out.  In  1882  these  were 
further  added  to  by  Mr.  W.  H.  Power's  report  concerning 
a  series  of  cases  in  Central  London.  That  report  was  re- 
markable for  the  introduction  of  a  new  feature,  viz.,  the 
evidence  produced  in  favour  of  the  infection  of  milk  from 
some  disease  of  the  cow.  The  Medical  Department  of  the 
Local  Government  Board  from  that  time  took  up  a  position 
of  suspended  judgment  concerning  the  belief  hitherto 
credited  that  milk  could  only  be  infected  by  human  scarlet 
fever.  In  1886  there  was  a  remarkable  epidemic  in  Mary- 
lebone,  and  the  theory  was  suggested  by  Dr.  Klein  and 
Mr.  Power  that  the  cow  from  which  the  milk  was  derived 
suffered  from  scarlet  fever. 

Into  the  extensive  controversy  which  raged  round  "  the 
Hendon  disease,"  as  it  was  called,  affecting  the  cows  supply- 
ing the  Marylebone  milk,  we  cannot  here  enter.  It  will  be 
sufficient  to  say  that  a  long  discussion  took  place  as  to 
whether  or  not  this  Hendon  disease  was  or  was  not  scarlet 
fever.  The  difficulty  of  course  largely  arose  from  the  fact 
before  mentioned  that  we  do  not  at  present  know  the  specific 


BACTERIA    IN  FOODS  203 

micro-organism  of  scarlet  fever.  The  Agricultural  Depart- 
ment supported  the  view  of  Professor  Crookshank  that  the 
cow  disease  at  Hendon  was  cowpox,  and  Professor  Axe 
further  pointed  out  that  there  was  evidence  of  the  Hendon 
milk  having  been  contaminated  with  human  scarlet  fever. 
Whichever  conclusion  was  adopted,  all  were  agreed  upon 
one  point,  viz.,  that  the  disease  had  been  conveyed  from 
Hendon  to  persons  in  Marylebone  by  means  of  the  milk. 

Mr.  Ernest  Hart  in  1897  published  a  very  large  number 
of  records  of  scarlatinal  milk  infection  from  all  parts  of  the 
country,  and  though  the  cause  of  the  disease  is  obscure, 
there  is  now  no  doubt  that  it  may  be  and  is  conveyed  by 
means  of  milk. 

Other  Diseases  Conveyed  by  Milk.  In  addition  to  the 
above,  there  are  other  diseases  spread  by  means  of  polluted 
milk.  From  time  to  time  exceptional  cases  have  occurred 
in  which  a  disease  like  anthrax  has  been  spread  by  this 
means.  But  it  is  not  to  such  rare  cases  that  we  refer. 
There  are  two  very  common  diseases  in  which  milk  has  been 
proved  to  play  a  not  inconsiderable  part,  viz.,  thrush  and 
diarrhoea. 

The  mould  which  gives  rise  to  the  curd-like  patches  in 
the  throats  of  children,  and  which  is  known  as  Oidium 
albicans,  frequently  occurs  in  milk.  Soft  white  specks  are 
seen  on  the  tongue  and  mucous  membrane  of  the  cheeks 
and  lips,  looking  not  unlike  particles  of  milk  curd.  If  a 
scraping  be  placed  upon  a  glass  slide  with  a  drop  of  glycerine 
and  examined  by  means  of  the  microscope,  the  spores  and 
mycelial  threads  of  this  mould  will  be  seen.  The  spores  are 
oval,  and  possess  a  definite  capsule.  The  threads  are 
branched  and  jointed  at  somewhat  long  intervals.  Milk 
affords  an  excellent  medium  for  the  growth  of  this  parasite. 
Thus  undoubtedly  we  must  hold  milk  partly  responsible  for 
spreading  this  complaint.  Penicillium,  Aspergillus,  and 
Mucor  are  also  frequent  moulds  in  milk. 


204  BACTERIA 

Professor  MacFadyen  '  has  given  a  full  account  of  the 
ways  in  which  milk  becomes  pathogenic,  and  his  views  have 
received  further  support  from  Professor  Sheridan  Delepine, 
who  has  examined  more  than  one  hundred  samples  of  milk 
from  Liverpool  and  Manchester.  The  result  of  this  invest- 
igation has  been  that  milk  must  be  held  to  be  one  of  the 
most  potent  causes  of  the  summer  diarrhoea  of  children. 
Indeed,  a  bacillus  has  been  isolated  identical  with  one  which 
was  apparently  the  cause  of  this  complaint,  which  carries  off 
such  a  large  number  of  infants  every  summer.  It  resembles 
closely  the  Bacillus  coli  communis,  which  is  an  almost  con- 
stant inhabitant  of  the  alimentary  canal,  and  is  held  by 
many  bacteriologists  to  play,  especially  in  conjunction  with 
yeasts  and  other  saprophytic  organisms,  an  active  role  in  the 
intestine  of  man. 

In  a  recent  official  report 2  Dr.  Hope,  of  Liverpool,  states 
that  "  the  method  of  feeding  plays  a  most  important 
part  in  the  causation  of  diarrhoea;  when  artificial  feeding 
becomes  necessary,  the  most  scrupulous  attention  should  be 
paid  to  feeding-bottles."  Careless  feeding,  in  conjunction 
with  a  warm,  dry  summer,  invariably  results  in  a  high  death- 
rate  from  this  cause.  These  two  causes  interact  upon  each 
other.  A  warm  temperature  is  a  favourable  temperature  for 
the  growth  of  the  poisonous  micro-organism ;  a  dry  season 
affords  ample  opportunity  for  its  conveyance  through  the 
air.  Unclean  feeding-bottles  are  obviously  an  admirable 
nidus  for  these  injurious  bacteria,  for  in  such  a  resting-place 
the  three  main  conditions  necessary  for  bacterial  life  are  well 
fulfilled,  viz.,  heat,  moisture,  and  pabulum.  The  heat  is 
supplied  by  the  warm  temperature,  the  moisture  and  food 
by  the  dregs  of  milk  left  in  the  bottle;  and  the  dry  air 
assists  in  transit. 

1  Journal  of  Comparative  Pathology,  vol.  x.  (1897),  pp.  150-189. 

2  E.  W.  Hope,  M.D.,  D.Sc.,  Report  of  the  Health  of  Liverpool  during  1897, 
p.  40. 


BACTERIA    IN  FOODS  2O$ 

Before  passing  on  to  other  matters,  reference  must  be 
made  to  poisonous  products  other  than  bacteria  which 
occur  in  milk  and  set  up  ill-health.  Vaughan,  of  Michigan, 
pointed  out  at  the  London  Congress  of  Hygiene  in  1891 
that  he  had  separated  a  poisonous  alkaloid,  which  he  called 
tyrotoxicon.  This,  as  its  name  denotes,  was  a  toxic  or 
poisonous  substance,  probably  produced  by  some  form  of 
microbe.  It  may  be  taken  as  a  type  of  the  organic  chemi- 
cal substances  frequently  occurring  in  milk. 

METHODS    OF   PRESERVING   MILK 

From  the  somewhat  extensive  category  of  diseases  which 
may  be  milk-borne,  it  will  be  suitable  now  to  speak  of  some 
of  the  means  at  our  disposal  for  obtaining  and  preserving 
good,  pure  milk. 

We  considered  at  the  commencement  of  this  chapter  the 
most  frequent  channels  of  contamination.  If  these  be 
avoided  or  prevented,  and  if  the  milk  be  derived  from  cows 
in  good  health  and  well  kept,  the  risk  of  infection  is  re- 
duced to  a  minimum.  But  we  have  seen  that  much,  if  not 
most,  of  the  pollution  of  milk  arises  after  the  milking  pro- 
cess and  during  transit  and  storage  preparatory  to  use. 
Bacteria  are  so  ubiquitous  that  to  prevent  the  entrance  of 
any  at  all  is  almost  beyond  hope.  Can  anything  be  done  to 
prevent  their  multiplication  or  to  kill  them  in  the  milk  ? 
Fortunately  the  answer  is  in  the  affirmative. 

There  are  two  means  at  hand  to  secure  these  results. 
First,  we  may  add  to  the  milk  various  chemical  or  physical 
preservatives.  Borax  or  boric  acid,  formaldehyde,  salicylic 
acid,  and  other  chemical  bodies  are  used  for  this  purpose. 
The  commonest  of  these  is  that  named  first.  The  Food 
and  Drugs  Act  (Section  VI.,  1875)  permits  the  addition  of 
an  ingredient  not  injurious  to  health  if  the  same  is  required 
for  protection  or  preparation  of  the  article  in  question.  It 


UNIVERSITY 

OF  / 


206  BACTERIA 

is,  however,  a  difficult  matter  to  determine  what  amount  of 
boric  acid  is  injurious  to  health,  for  this  differs  widely  in 
different  persons.  It  has  been  laid  down  by  one  authority 
that  even  so  small  an  amount  as  one-tenth  per  cent,  might 
have  inconvenient  results,  owing  to  its  cumulative  effect. 
Formaldehyde  is  without  doubt  an  excellent  antiseptic,  and 
the  more  its  efficacy  becomes  known  so  much  the  more 
probably  will  it  be  used.  The  salicylates,  which  are  mild 
antiseptics,  have  long  been  used  as  preservatives.  These 
substances,  then,  can  be  added  to  milk  in  quantities  not 
recognisable  to  the  taste  (salicylic  acid  about  .75  grain,  and 
boracic  acid  .4  grain,  to  the  litre  of  milk).  They  will  ma- 
terially increase  the  time  that  milk  will  remain  sweet,  they 
will  prevent  a  number  of  micro-organisms  living  in  the  milk, 
and  will  inhibit  multiplication  of  others.1  Secondly,  it  is 
possible  very  perceptibly  to  remove  the  infectivity  of  milk 
by  filtration  and  temperature  variations. 

Filtration  has  been  practised  for  some  time  by  the  Copen- 
hagen Dairy  Company  and  by  Bolle,  of  Berlin.  The  filters 
used  consist  of  large  cylindrical  vessels  divided  by  horizontal 
perforated  diaphragms  into  five  superposed  compartments, 
of  which  the  middle  three  are  filled  with  fine  sand  of  three 
sizes.  At  the  bottom  is  the  coarsest  sand,  and  at  the  top 
the  finest.  The  milk  enters  the  lowest  compartment  by  a 
pipe  under  gravitation  pressure,  and  is  forced  upwards,  and 
finally  is  run  off  into  an  iced  cooler,  and  from  that  into 
the  distribution  cans.  By  this  means  the  number  of  bac- 
teria is  reduced  to  one-third.  The  difficulty  of  drying  and 
sterilising  enough  sand  to  admit  a  large  turnover  of  milk  is 
a  serious  one.  This,  in  conjunction  with  the  belief  that 

1  S.  Rideal  and  A.  G.  R.  Foulerton  conclude,  from  a  series  of  experiments, 
that  boric  acid  (1-2000)  and  formaldehyde  (1-50,000)  are  effective  preserva- 
tives for  milk  for  a  period  of  twenty-four  hours,  and  that  these  quantities  have 
no  appreciable  effect  upon  digestion  or  the  digestibility  of  foods  preserved  by 
them  {Public  Health,  May,  1899,  pp.  554-568). 


BACTERIA    IN  FOODS 


207 


filtration  removes  some  of  the  essential  nutritive  elements 
of  milk,  has  caused  the  process  to  be  but  little  adopted. 
Dr.  Seibert  states  that  if  milk  be  filtered  through  half  an 
inch  of  compressed  absorbent  cotton,  seven-eighths  of  the 
contained  bacteria  will  be  removed,  and  a  second  filtration 
will  further  reduce  the  number  to  one-twentieth.  One 
quart  of  milk  may  thus  be  filtered  in  fifteen  minutes. 

The  common  methods  now  in  vogue  for  the  protection  of 
milk  are  based  upon  gcrmicidal  temperatures.  Low  temper- 
atures, it  is  true,  do  not  easily  destroy  life,  but  they  have  a 
most  beneficial  effect  upon  the  keeping  quality  of  milk.  At 
the  outset  of  the  process  of  cooling,  strong  currents  of  air  are 
started  in  the  milk-can,  which  act  mechanically  as  deodor- 
isers. But  if  the  temperature  be  lowered  sufficiently,  the 
contained  bacteria  become  inactive  and  torpid,  and  event- 
ually are  unable  to  multiply  or  produce  their  characteristic 
fermentations.  At  about  50°  F.  (10°  C.)  the  activity  ceases, 
and  at  temperatures  of  45°  F.  (7°  C.)  and  39°  F.  (4°  C.) 
organisms  are  deprived  of  their  injurious  powers.  If  it  hap- 
pens that  the  milk  is  to  be  conveyed  long  distances,  then 
even  a  lower  temperature  is  desirable.  The  most  important 
point  with  regard  to  the  cooling  of  milk  is  that  it  should 
take  place  quickly.  Various  kinds  of  apparatus  are  effective 
in  accomplishing  this.  Perhaps  those  best  known  are  Law- 
rence's cooler  and  Pfeiffer's  cooler,  the  advantage  of  the 
latter  being  that  during  the  process  the  milk  is  not  exposed 
to  the  air.  It  must  not  be  forgotten  that  cooling  processes 
are  not  sterilising  processes.  They  do  not  necessarily  kill 
bacteria;  they  only  inhibit  activity,  and  under  favourable 
circumstances  the  torpid  bacteria  may  again  acquire  their 
injurious  faculties.  Hence  during  the  cooling  of  milk 
greater  care  must  be  taken  to  prevent  aerial  contamination 
than  is  necessary  during  the  process  of  sterilising  milk.  No 
cooling  whatever  should  be  attempted  in  the  stable;  but, 
on  the  other  hand,  there  should  be  no  delay.  Climate 


208  BACTERIA 

makes  little  or  no  difference  to  the  practical  desirability  of 
cooling  milk,  yet  it  is  obvious  that  less  cooling  will  be  re- 
quired in  the  cold  season. 

We  now  come  to  the  protective  processes  known  as  steril- 
isation and  pasteurisation.  As  we  have  already  seen,  steril- 
isation indicates  a  complete  and  final  destruction  of  bacteria 
and  their  spores.  As  applied  to  methods  of  preserving  milk, 
sterilisation  means  the  use  of  heat  at,  or  above,  boiling- 
point,  or  boiling  under  pressure.  This  may  be  applied  in 
one  application  of  one  to  two  hours  at  250°  F.,  or  it  may 
be  applied  at  stated  intervals  at  a  lower  temperature.  The 
milk  is  sterilised — that  is  to  say,  contains  no  living  germs- 
is  altered  in  chemical  composition,  and  is  also  boiled  or 
"  cooked,"  and  hence  possesses  a  flavour  which  to  many 
people  is  unpalatable. 

Now,  such  a  radical  alteration  is  not  necessary  in  order  to 
secure  non-infectious  milk.  The  bacteria  causing  the  dis- 
eases conveyable  by  milk  succumb  at  much  lower  temper- 
atures than  the  boiling-point.  Advantage  is  taken  of  this 
in  the  process  known  as  "  pasteurisation."  By  this  method 
the  milk  is  heated  to  167-185°  F.  (75-85°  C.).  Such  a 
temperature  kills  harmful  microbes,  because  75°  C.  is  de- 
cidedly above  their  average  thermal  death-point,  and  yet 
the  physical  changes  in  the  milk  are  practically  nil,  because 
85°  C.  does  not  relatively  approach  the  boiling-point. 
There  is  no  fixed  standard  for  pasteurisation,  except  that 
it  must  be  above  the  thermal  death-point  of  pathogenic 
bacteria,  and  yet  below  the  boiling-point.  As  a  matter  of 
fact,  158°  F.  (70°  C.)  will  kill  all  souring  bacteria  as  well  as 
disease-producing  organisms  found  in  milk.  If  the  milk  is 
kept  at  that  temperature  for  ten  or  fifteen  minutes,  we  say 
it  has  been  "  pasteurised."  If  it  has  been  boiled,  with  or 
without  pressure,  for  half  an  hour,  we  say  it  has  been 
"  sterilised."  The  only  practical  difference  in  the  result  is 
that  sterilised  milks  have  a  better  keeping  quality  than 


BACTERIA    IN  FOODS  2OQ 

pasteurised,  for  the  simple  reason  that  in  the  latter  some 
living  germs  have  been  unaffected. 

Sterilisation  may  of  course  be  carried  out  in  a  variety  of 
modifications  of  the  two  chief  ways  above  named.  When 
the  process  is  to  be  completed  in  one  event  an  autoclave  is 
used,  in  order  to  obtain  increased  pressure  and  a  higher 
temperature.  Milk  so  treated  is  physically  changed  in 
greater  degree  than  in  the  slower  process.  The  slow  or 
intermittent  method  is,  of  course,  based  on  Tyndall's  dis- 
covery that  actively  growing  bacteria  are  more  easily  killed 
than  their  spores.  The  first  sterilisation  kills  the  bacteria, 
but  leaves  their  spores.  By  the  time  of  the  second  appli- 
cation the  spores  have  developed  into  bacteria,  which  in 
turn  are  killed  before  they  can  sporulate. 

The  methods  of  pasteurisation  are  continually  being 
modified  and  improved,  especially  in  Germany  and  America. 
Most  of  the  variations  in  apparatus  may  be  classed  under 
two  headings.  There  are,  first,  those  in  which  a  sheet  of 
milk  is  allowed  to  flow  over  a  surface  heated  by  steam  or 
hot  water.  This  may  be  a  flat,  corrugated  surface  or  a 
revolving  cylinder.  The  milk  is  then  passed  into  coolers. 
Secondly,  milk  is  pasteurised  by  being  placed  in  reservoirs 
surrounded  by  an  external  shell  containing  hot  water  or 
steam.  Dr.  A.  L.  Russell 1  has  described  one  apparatus 
consisting  of  a  pasteuriser,  a  water-cooler,  and  an  ice-cooler. 
The  pasteuriser  is  heated  by  hot  water  in  the  outside  case- 
ment. To  equalise  rapidly  the  temperature  of  the  water 
and  milk  a  series  of  agitators  must  be  used.  These  are 
suspended  on  movable  rods,  and  hang  vertically  in  the  milk 
and  vater  chambers.  By  this  ingenious  arrangement  the 
heat  is  diffused  rapidly  throughout  the  whole  mass,  and  as 
the  temperature  of  the  milk  reaches  the  proper  point  the 
steam  is  shut  off,  and  the  heat  of  the  whole  body  of  water 
and  milk  will  remain  constant  for  the  proper  length  of  time. 

1  Report  from  Wisconsin  Agricultural  Experiment  Station,  1896. 


210  BACTERIA 

The  somewhat  difficult  problem  of  drawing  off  the  pasteur- 
ised milk  from  the  vat  without  reinfecting  it  by  contact  with 
the  air  is  solved  by  placing  a  valve  inside  the  chamber,  and 
by  means  of  a  pipe  leading  the  pasteurised  milk  directly  and 
rapidly  into  the  coolers.  These  are  of  two  kinds,  which  may 
be  used  separately  or  conjointly.  In  one  set  of  cylinders 
there  is  cold  circulating  water,  in  the  other  finely  crushed  ice. 

Domestic  pasteurisation  can  be  accomplished  readily  by 
heating  the  milk  in  vessels  in  a  water-bath  raised  to  the 
required  temperature  for  half  an  hour. 

Without  entering  into  a  long  discussion  upon  the  various 
methods  adopted,  we  may  summarise  some  of  the  chief 
essential  conditions.  It  need  scarcely  be  said  that  the 
operation  must  be  efficiently  conducted,  and  in  such  a  way 
as  to  maintain  absolute  control  over  the  time  and  temper- 
ature. The  apparatus  should  be  simple  enough  to  be  easily 
cleansed,  sterilised,  and  economical  in  use.  Arrangements 
must  always  be  made  to  protect  the  milk  from  reinfection 
during  and  after  the  process.  The  entire  preparation  of  the 
milk  for  market  may  be  summed  up  in  four  items : 

1.  Pasteurisation  in  heat  reservoir. 

2.  Rapid  cooling  in  water-  or  ice-coolers. 

3.  All  cans,  pails,  bottles,  and  other  utensils  to  be  thoroughly 
sterilised  in  steam. 

4.  The  prepared  milk  must  be  placed  in  sterilised  bottles  and 
sealed  up. 

The  quality  of  the  milk  to  be  pasteurised  is  an  important 
point.  All  milks  are  not  equally  suited  for  this  purpose, 
and  those  containing  a  large  quantity  of  contamination, 
especially  of  spores,  are  distinctly  unsuitable.  Such  milks, 
to  be  purified,  must  be  sterilised.  Dr.  Russell  has  laid 
down  a  standard  test  for  the  degree  of  contamination  which 
may  be  corrected  by  pasteurisation  by  estimating  the  degree 
of  acidity,  a  low  acidity  (e.  g.,  0.2  per  cent.)  usually  in- 


BACTERIA   IN  FOODS  211 

dicating  a  smaller  number  of  spore-bearing  germs  than  that 
which  contains  a  high  percentage  of  acid. 

Lastly,  while  the  heating  process  is  of  course  the  essential 
feature  of  efficient  pasteurisation,  it  must  not  be  forgotten 
that  rapid  and  thorough  cooling  is  almost  equally  import- 
ant. As  we  have  seen,  pasteurisation  differs  from  complete 
sterilisation  in  that  it  leaves  behind  a  certain  number  of 
microbes  or  their  spores.  Cooling  inhibits  the  germination 
and  growth  of  this  organismal  residue.  If  after  the  heating 
process  the  milk  is  cooled  and  kept  in  a  refrigerator,  it  will 
probably  keep  sweet  from  three  to  six  days,  and  may  do  so 
for  three  weeks. 

Before  leaving  this  subject  we  may  glance  for  a  moment 
at  the  bacterial  results  of  pasteurisation  and  sterilisation. 
The  chief  two  of  these  are  the  enhanced  keeping  quality 
and  the  removal  of  disease-producing  germs.  The  former 
is  due  in  part  to  the  latter,  and  also  to  the  removal  of  the 
lactic  acid  and  other  fermentative  bacteria.  As  a  general 
rule  these  bacteria  do  not  produce  spores,  and  hence  they 
are  easily  annihilated  by  pasteurisation.  True,  a  number 
of  indifferent  bacteria  are  untouched,  and  also  some  of  the 
peptonising  species.  The  cooling  itself  contributes  to  the 
increased  keeping  power  of  the  milk,  especially  in  transit  to 
the  consumer. 

Pasteurised  milks  have  the  following  three  economical  and 
commercial  advantages  over  sterilised  milks,  namely,  they 
are  more  digestible,  the  flavour  is  not  altered,  and  the  fat 
and  lact-albumen  are  unchanged.  Professor  Hunter  Stew- 
art, of  Edinburgh,  about  two  years  ago,  compiled  from  a 
number  of  experiments  the  following  instructive  and  com- 
prehensive table  (page  212). 

It  will  be  admitted  that  this  table  exhibits  much  in  favour 
of  pasteurisation ;  yet  the  crucial  test  must  ever  be  the  effect 
upon  pathogenic  bacteria.  Fliigge  has  conducted  a  series 
of  experiments  upon  the  destruction  of  bacteria  in  milk,  and 


212 


BACTERIA 


he  states  that  a  temperature  of  158°  F.  (70°  C.)  maintained 
for  thirty  minutes  will  kill  the  specific  organisms  of  tubercle, 
diphtheria,  typhoid,  and  cholera.  MacFadyen  and  Hewlett 
have  demonstrated,1  by  sudden  alternate  heating  and  cool- 
ing, that  70°  C.  maintained  for  half  a  minute  is  generally 
sufficient  to  kill  suppurative  organisms  and  such  virulent 
types  of  pathogenic  bacteria  as  Bacillus  diphtheric?,  B. 
typhosus,  and  B.  tuberculosis. 


No.  of 
Experi- 
ments. 

Average  No. 
of  Microbes 
per  cc.  in 
Milk  before 
Treatment. 

Temperature 
and  Dura- 
tion of 
Pasteurisa- 
tion in 
Minutes. 

No.  of  Microbes 
per  cc.  in 
Pasteurised  Milk 
after  24  Hours. 

Soluble 
Albumen 
in 
Fresh  Milk, 
per  cent. 

Soluble 
Albumen 
in 
Pasteurised 
Milk, 
per  cent. 

Taste  of 
Pasteurised  Milk. 

5 

136,262 

10'  60°  C. 

1722  average 

0.423 

0.418 

Unaffected 

4 

53,656 

30'  60°  C. 

i  sterile 

0.435 

0.427 

'  i 

3  averaged  955 

12 

78,562 

10'  65°  C. 

6  sterile 

0.395 

0.362 

Not    apprecia- 

6 averaged  686 

bly  affected 

12 

132,833 

30'  65°  C. 

9  sterile 

0.395 

0.333 

'  ' 

3  averaged  233 

13 

49,867 

10'  70°  C. 

sterile 

0.422 

0.269 

Slightly  boiled 

9 

38,320 

30'  70°  C. 

* 

0.421 

0.253 

*  * 

2 

77,062 

10'  75°  C. 

1 

0.38 

0.07 

Boiled 

3 

48,250 

30'  75°  C. 

1 

0.38 

0.05 

" 

i 

1,107,000 

10'  80°  C. 

1 

0.375 

o.oo 

" 

i 

1,107,000 

30'  80°  C. 

* 

0.375 

o.oo 

*  * 



Respecting  the  numerical  diminution  of  microbes  brought 
about  by  pasteurisation  and  sterilisation,  respectively,  we 
may  take  the  following  two  sets  of  experiments.  Dr.  N. 
L.  Russell8  tabulates  the  immediate  results  of  pasteurisation 
as  follows: 


UNPASTEURISED. 


Minimum. 

Maximum. 

Average. 

Minimum. 

Maximum. 

Average. 

Full    cream 

milk  

25,300 

18,827,000 

3,674,000 

O 

37,500 

6,140 

Cream,  25$. 

425,OOO 

32,8OO,OOO 

8,7OO,OOO 

O 

57,000 

24,250 

PASTEURISED. 


1  Jenner  Institute  of  Preventive  Medicine  (First  Series  Transactions). 
*  Centralblatt  fur  Bakleriologie,  etc.,  II.  Abteilung. 


BACTERIA   IN  FOODS  21$ 

As  regards  the  later  effect  of  the  process,  he  states  that  in 
fifteen  samples  of  pasteurised  milk  examined  from  Novem- 
ber to  December  nine  of  them  revealed  no  organisms,  or  so 
few  that  they  might  almost  be  regarded  as  sterile ;  in  those 
samples  examined  after  January  the  lowest  number  was  100 
germs  per  cc.,  while  the  average  was  nearly  5000.  With 
the  pasteurised  cream  a  similar  condition  was  to  be  ob- 
served. 

Dr.  Hewlett 1  defines  pasteurisation  briefly  as  heating  the 
milk  to  68°  C.  for  twenty  or  thirty  minutes,  and  this  treat- 
ment he  quotes  as  destroying  99.75  per  cent,  of  the  total 
number  of  organisms.  Bitter's  table  of  results  at  158°  F. 
bears  out  the  same : 

BEFORE  PASTEURISATION.          AFTER  PASTEURISATION. 
No.  of  Bacteria  in  10  Drops.     No.  of  Bacteria  in  10  Drops. 

I 102,600 2 — 3 

2 25 1,600 30— 40 

3 25,000 3—5 

4 37,5°° 2~5 

5 94,000 2 

BACTERIA   IN    MILK   PRODUCTS 

Cream  is  generally  richer  in  bacteria  than  milk.  Set  cream 
contains  more  bacteria  than  separated  cream,  but  germs  are 
abundant  in  both.  Yet  whilst  it  is  true  that  cream  contains 
a  large  number  of  bacteria,  it  must  be  pointed  out  that  the 
butter  fat  in  cream  is  a  less  suitable  food  for  organisms  than 
is  the  case  with  milk.  Hence  the  fermentative  changes  set 
up  in  cream  are  of  less  degree  than  in  milk,  particularly  so 
if  separated  from  the  milk.  Butter-milk  and  whey  vary 
much  in  their  bacterial  content.  Butter  necessarily  follows 
the  standard  of  the  cream.  But  as  the  butter  fat  is  not 
well  adapted  for  bacterial  food,  the  number  of  bacteria  in 

1  A  Manual  of  Bacteriology,  Clinical  and  Applied,  p.  397. 


214  BACTERIA 

butter  is  usually  less  than  in  cream.1  Moreover,  they  are 
soon  reduced  both  in  quality  and  quantity.  Butter  ex- 
amined after  it  is  several  months  old  is  often  found  to  be 
almost  free  from  germs;  yet  in  the  intervening  period  a 
variety  of  conditions  are  set  up  directly  or  indirectly  through 
bacterial  action. 

Rancid  butter  is  partly  due  to  organisms.  Putrid  butter 
is  caused,  according  to  Jensen,  by  various  putrefactive  bac- 
teria, one  form  of  which  is  named  Bacillus  foetidus  lactis* 
This  organism  is  killed  at  a  comparatively  low  temperature, 
and  is  therefore  completely  removed  by  pasteurisation. 
Ill- flavoured  butter  may  be  due  to  germs  or  an  unsuitable 
diet  of  the  cow  and  a  retention  of  the  bad  quality  of  the 
resulting  milk.  Lardy  and  oily  butters  have  been  investi- 
gated by  Storch  and  Jensen  and  traced  to  bacteria.  Lastly, 
bitter  butter  occasionally  occurs,  and  is  due  to  fermentative 
changes  in  the  milk.  Butter  may  also  contain  pathogenic 
bacteria,  like  tubercle.  The  B.  coli  can  live  for  one  month 
in  butter. 

Cheese  suffers  from  very  much  the  same  kind  of  "  dis- 
eases "  as  butter,  except  that  chromogenic  conditions  occur 
more  frequently.  The  latter  are,  under  certain  circum- 
stances, more  the  result  of  chemical  than  bacterial  action. 
Most  of  the  troubles  in  cheese  originate  in  the  milk. 

Method  of  Examination  of  Butter.  Several  grams  of  the 
butter  should  be  placed  in  a  large  test-tube,  which  is  then 
two-thirds  filled  with  sterilised  water  and  placed  in  a  water- 
bath  at  about  45°  C.  until  the  butter  is  completely  melted. 
A  small  quantity  may  then  be  added  to  gelatine  or  agar  and 
plated  out  on  Petri  dishes  or  in  flat-bottomed  flasks  in  the 
usual  way.  After  which  the  tube  may  be  well  shaken  and 
returned  to  the  bath  inverted.  In  the  space  of  twenty  or 
thirty  minutes  the  butter  has  separated  from  the  water  with 

1  Hewlett  asserts  that  butter  may  contain  from  two  to  forty-seven  millions  of 
bacteria  per  gram. 


BACTERIA    IN  FOODS  21$ 

which  it  has  been  emulsified.  It  is  then  placed  in  the  cold 
to  set.  The  water  may  be  now  either  centrifugalised  or 
placed  in  sedimentation  flasks,  and  the  deposit  examined 
for  bacteria. 

The  Uses  of  Bacteria  in  Dairy  Produce.  In  considering  the 
relation  of  bacteria  to  milk  we  found  that  many  of  the 
species  present  were  injurious  rather  than  otherwise,  and 
when  we  come  to  consider  bacteria  in  dairy  products,  like 
butter  and  cheese,  we  find  that  the  dairyman  possesses  in 
them  very  powerful  allies.  Within  recent  years  almost  a 
new  industry  has  arisen  owing  to  the  scientific  application 
of  bacteriology  to  dairy  work. 

As  a  preliminary  to  butter-making  the  general  custom  in 
most  countries  is  to  subject  the  cream  to  a  process  of 
"  ripening."  As  we  have  seen,  cream  in  ordinary  dairies 
and  creameries  invariably  contains  some  bacteria,  a  large 
number  of  which  are  in  no  sense  injurious.  Indeed,  it  is  to 
these  bacteria  that  the  ripening  and  flavouring  processes 
are  due.  They  are  perfectly  consistent  with  the  production 
of  the  best  quality  of  butter.  The  aroma  of  butter,  as  we 
know,  controls  in  a  large  measure  its  price  in  the  market. 
This  aroma  is  due  to  the  decomposing  effect  upon  the  con- 
stituents of  the  butter  of  the  bacteria  contained  in  the 
cream.  In  the  months  of  May  and  June  the  variety  and 
number  of  these  types  of  bacteria  are  decidedly  greater  than 
in  the  winter  months,  and  this  explains  in  part  the  better 
quality  of  the  butter  at  these  seasons.  As  a  result  of  these 
ripening  bacteria  the  milk  becomes  changed  and  soured,  and 
slightly  curdled.  Thus  it  is  rendered  more  fit  for  butter- 
making,  and  acquires  its  pleasant  taste  and  aroma.  It  is 
then  churned,  after  which  bacterial  action  is  reduced  to  a 
minimum  or  is  absent  altogether.  Sweet-cream  butter  lacks 
the  flavour  of  ripened  or  sour-cream  butter.  The  process  is 
really  a  fermentation,  the  ripening  bacteria  acting  on  each 
and  all  of  the  constituents  of  the  milk,  resulting  in  the 


2l6  BACTERIA 

production  of  various  bye-products.  This  fermentation  is  a 
decomposition,  and  just  as  we  found  when  discussing  fer- 
mentation, so  here  also  the  action  is  beneficial  only  if  it  is 
stopped  at  the  right  moment.  If,  for  example,  instead  of 
being  stopped  on  the  second  day,  it  is  allowed  to  continue 
for  a  week,  the  cream  will  degenerate  and  become  offensive, 
and  the  pleasant  ripening  aroma  will  be  changed  to  the 
contrary. 

Bacteriologists  have  demonstrated  that  butters  possessing 
different  flavours  have  been  ripened  by  different  species  of 
bacteria.  Occasionally  one  comes  across  a  dairy  which 
seems  to  be  impregnated  with  bacteria  that  improve  cream 
and  flavour  well.  In  other  cases  the  contrary  happens,  and 
a  dairy  becomes  impregnated  with  a  species  having  deleter- 
ious effects  upon  its  butter.  This  species  may  arise  from 
unclean  utensils  and  dairying,  from  disease  of  the  cow,  or 
from  a  change  in  the  cow's  diet.  Thus  it  comes  about  that 
the  butter-maker  is  not  always  able  to  depend  upon  good 
ripening  for  his  cream.  At  other  times  he  gets  ripening  to 
occur,  but  the  flavour  is  an  evil  one,  and  the  results  corre- 
spond. It  may  be  bitter  or  tainted,  and  just  as  certainly  as 
these  flavours  develop  in  the  cream,  so  is  it  certain  that  the 
butter  will  suffer.  Fortunately  the  bacterial  content  of  the 
cream  is  generally  either  favourable  or  indifferent  in  its 
action.  Thus  it  comes  about  that  the  custom  is  to  allow 
the  cream  simply  to  ripen,  so  to  speak,  of  its  own  accord,  in 
a  vat  exposed  to  the  influence  of  any  bacteria  which  may 
happen  to  be  around.  This  generally  proves  satisfactory, 
but  it  has  the  great  disadvantage  of  being  indefinite  and  un- 
certain. Occasionally  it  turns  out  wholly  unsatisfactory, 
and  results  in  financial  loss. 

There  are  various  means  at  our  command  for  improving 
the  ripening  process.  Perfect  cleanliness  in  the  entire 
manipulation  necessary  in  milking  and  dairying,  combined 
with  freedom  from  disease  in  the  milch  cows,  will  carry  us  a 


BACTERIA    IN  FOODS 


217 


long  way  on  the  road  towards  a  good  cream-ripening.  Re- 
cently, however,  a  new  method  has  been  introduced,  largely 
through  the  work  and  influence  of  Professor  Storch  in  Den- 
mark, which  is  based  upon  our  new  knowledge  respecting 
bacterial  action  in  cream-ripening.  We  refer  to  the  artificial 
processes  of  ripening  set  up  by  the  addition  of  pure  cultures 
of  favourable  germs*  If  a  culture  of  organisms  possessing 
the  faculty  of  producing  in  cream  a  good  flavour  be  added 
to  the  sweet  cream,  it  is  clear  that  advantage  will  accrue. 
This  simple  plan  of  starting  any  special  or  desired  flavour 
by  introducing  the  specific  micro-organism  of  that  flavour 
may  be  adopted  in  two  or  three  different  ways.  If  cream 
be  inoculated  with  a  large,  pure  culture  of  some  particular 
kind  of  bacteria,  this  species  will  frequently  grow  so  well 
and  so  rapidly  that  it  will  check  the  growth  of  the  other 
bacteria  which  were  present  in  the  cream  at  the  commence- 
ment and  before  the  starter  was  added.  That  is,  per- 
haps, the  simplest  method  of  adding  an  artificial  culture. 
But  secondly,  it  will  be  apparent  to  those  who  have  followed 
us  thus  far,  that  if  the  cream  is  previously  pasteurised  at  70° 
C.  these  competing  bacteria  will  have  been  mostly  or  entirely 
destroyed,  and  the  pure  culture,  or  starter,  will  have  the 
field  to  itself.  There  is  a  third  modification,  which  is  some- 
times termed  ripening  by  natural  starters.  A  natural 
starter  is  a  certain  small  quantity  of  cream  taken  from  a 
favourable  ripening — from  a  clean  dairy  or  a  good  herd — 
and  placed  aside  to  sour  for  two  days  until  it  is  heavily  im- 
pregnated with  the  specific  organism  which  was  present  in 
the  whole  favourable  stock  of  which  the  natural  starter  is 
but  a  part.  It  is  then  added  to  the  new  cream  the  favour- 
able ripening  of  which  is  desired.  Of  the  species  which 
produce  good  flavours  in  butter  the  majority  are  found  to 

1  Such  pure  cultures  for  such  purposes  are  in  the  United  States  termed 
"  starters,"  because  they  start  the  process  of  special  ripening.  For  the  sake  of 
convenience  the  term  will  be  used  here. 


2l8  BACTERIA 

< 

be  members  of  the  acid-producing  class ;  but  probably  the 

flavour  is  not  dependent  upon  the  acid.  Moreover,  the 
aroma  of  good  ripening  is  also  probably  independent  of 
the  acid  production. 

Of  all  the  methods  of  ripening — natural  ripening,  the 
addition  of  natural  starters,  the  addition  of  pure  cultures 
with  or  without  pasteurisation — there  can  be  no  doubt  that 
pure  culture  after  pasteurisation  is  the  most  accurate  and 
dependable.  The  use  of  natural  starters  is  a  method  in 
the  right  direction;  yet  it  is,  after  all,  a  mixed  culture, 
and  therefore  not  uniform  in  action.  In  order  to  obtain 
the  best  results  with  the  addition  of  pure  cultures,  Professor 
Russell  has  made  the  following  recommendations: 

1.  The  dry  powder  of  the  pure  culture  must  be  added  to 
a  small  amount  of  milk  that  has  been  first  pasteurised,  in 
order  to  develop  an  active  growth  from  the  dried  material. 

2.  The  cream  to  be  ripened  must  first  be  pasteurised,  in 
order  to  destroy  the  developing  organisms  already  in  it,  and 
thus  be  prepared  for  the  addition  of  the  pure  culture. 

3.  The  addition  of  the  developing    starter  to  the  pas- 
teurised  cream  and  the  holding  of   the   cream  at  such  a 
temperature  as  will  readily  induce  the  best  development  of 
flavour. 

4.  The  propagation  of  the  starter  from  day  to  day.     A 
fresh  lot  of  pasteurised   milk  should   be  inoculated   daily 
with  some  of  the  pure  culture  of  the  previous  day,  not  the 
ripening  cream   containing  the  culture.     In  this  way  the 
purity  of  the  starter  is  maintained  for  a  considerable  length 
of  time.     Those  starters  are  best  which  grow  rapidly  at  a 
comparatively  low  temperature  (60-75°  F.),  which  produce 
a  good  flavour,  and  which  increase  the  keeping  qualities  of 
the  butter.     Now,  whilst  it  is  true  that  the  practice  of  using 
pure  cultures  in  this  way  is  becoming  more  general,  very 
few  species  have  been  isolated  which  fulfil  all  the  desirable 
qualities  above  mentioned.     In  America  starters  are  pre- 


BACTERIA   IN  FOODS  219 

f erred  which  yield  a  "  high  "  flavour,  whereas  in  Danish 
butter  a  mild  aroma  is  commoner.  In  England  as  yet 
very  little  has  been  done,  and  that  on  an  experimental  scale 
rather  than  a  commercial  one.1  In  1891  it  appears  that  only 
4  per  cent,  of  the  butter  exhibited  at  the  Danish  butter 
exhibitions  was  made  from  pasteurised  cream  plus  a  culture 
starter;  but  in  1895,  86  per  cent,  of  the  butter  was  so  made. 
Moreover,  such  butter  obtained  the  prizes  awarded  for  first- 
class  butter  with  preferable  flavour.  Different  cultures  will, 
of  course,  yield  different  flavoured  butter.  If  we  desire,  say, 
a  Danish  butter,  then  some  species  like  "  Hansen's  Danish 
Starter  "  would  be  added  ;  if  we  desire  an  American  butter, 
we  should  use  a  species  like  that  known  as  "  Conn's  Bacil- 
lus, No.  41."  But  whilst  these  are  two  common  types, 
they  are  not  the  only  suitable  and  effective  starters.  On 
certain  farms  in  England  there  are  equally  good  cultures, 
which,  placed  under  favourable  temperatures  in  new  cream, 
would  immediately  commence  active  ripening. 

Professor  H.  W.  Conn,  who,  with  Professor  Russell,  has 
done  so  much  in  America  for  the  advancement  of  dairy 
bacteriology,  reports  3  a  year's  experience  with  the  bacillus 
to  which  reference  has  been  made,  and  which  is  termed  No. 
41.  It  was  originally  obtained  from  a  specimen  of  milk 
from  Uruguay,  South  America,  which  was  exhibited  at  the 
World's  Fair  in  Chicago,  and  proved  the  most  successful 
flavouring  and  ripening  agent  among  a  number  of  cultures 
that  were  tried.  The  conclusions  arrived  at  after  a  con- 
siderable period  of  testing  and  experimentation  appear  to  be 
on  the  whole  satisfactory.  A  frequent  method  of  testing 

1  The  Essex  County  Council  is  one  of  the  few  public  bodies  in  England  which 
have  undertaken  pioneer  work  in  this  department  of  industry.  Under  the 
leadership  of  Mr.  David  Houston,  a  course  of  elementary  instruction  in  dairy 
bacteriology  as  applied  to  modern  dairy  practice  is  given  in  the  County  Bio- 
logical Laboratory  at  Chelmsford. 

9  Report  of  Storr's  Agricultural  Experiments  Station,  State  of  Connecticut, 
1895. 


220  BACTERIA 

has  been  to  divide  a  certain  quantity  of  cream  into  two 
parts,  one  part  inoculated  with  the  culture  and  the  other 
part  left  uninoculated.  Both  have  then  been  ripened  under 
similar  conditions,  and  churned  in  the  same  way ;  the  differ- 
ences have  then  been  noted.  It  is  interesting  to  know  that, 
as  a  result  of  the  year's  experience,  creameries  have  been 
able  to  command  a  price  varying  from  half  a  cent  to  two 
cents  a  pound  more  for  the  "  culture  "  butters  than  for  the 
uninoculated  butters.  The  method  advised  in  using  this 
pure  culture  is  to  pasteurise  (by  heating  at  155°  F.)  six 
quarts  of  cream,  and  after  cooling  to  dissolve  in  this  cream 
the  pellet  containing  bacillus  No.  41.  The  cream  is  then 
set  in  a  warm  place  (70°  F.),  and  the  bacillus  is  allowed  to 
grow  for  two  days,  and  is  then  inoculated  into  twenty-five 
gallons  of  ordinary  cream.  This  is  allowed  to  ripen  as 
usual,  and  is  then  used  as  an  infecting  culture,  or  "  starter," 
in  the  large  cream  vats  in  the  proportion  of  one  gallon  of 
infecting  culture  to  twenty-five  gallons  of  cream,  and  the 
whole  is  ripened  at  a  temperature  of  about  68°  F.  for  one 
day.  The  cream  ripened  by  this  organism  needs  to  be 
churned  at  a  little  lower  temperature  (say  52°-54°  F.)  but  to 
be  ripened  at  a  little  higher  temperature  than  ordinary  cream 
to  produce  the  best  results.  Cream  ripened  with  No.  41 
has  its  keeping  power  much  increased,  and  the  body  or 
grain  of  the  butter  is  not  affected.  More  than  two  hundred 
creameries  in  America  used  this  culture  during  1895,  and 
Professor  Conn  reports  that  this  has  proved  that  its  use  for 
the  production  of  flavour  in  butter  is  feasible  in  ordinary 
creameries  and  in  the  hands  of  ordinary  butter-makers  pro- 
vided they  will  use  proper  methods  and  proper  discretion. 

Bacteria  in  Cheese -making.  The  cases  where  it  has  been 
possible  to  trace  bacterial  disease  to  the  consumption  of 
butter  and  cheese  have  been  rare.  Notwithstanding  this 
fact,  it  must  not  be  supposed  that  therefore  cheese  contains 
few  or  no  bacteria.  On  the  contrary,  for  the  making  of 


BACTERIA   IN  FOODS  221 

cheese  bacteria  are  not  only  favourable,  but  actually  essen- 
tial, for  in  its  manufacture  the  casein  of  the  milk  has  to  be 
separated  from  the  other  products  by  the  use  of  rennet,  and 
is  then  collected  in  large  masses  and  pressed,  forming  the 
fresh  cheese.  In  the  course  of  time  this  undergoes  ripening, 
which  develops  the  peculiar  flavours  characteristic  of  cheese, 
and  upon  which  its  whole  value  depends. 

We  have  said  that  the  casein  is  separated  by  the  addition 
of  rennet,  which  has  the  power  of  coagulating  the  casein. 
But  this  precipitation  may  also  be  accomplished  by  allowing 
acid  to  develop  in  the  milk  until  the  casein  is  precipitated, 
as  in  some  sour-milk  or  cottage  cheeses.  The  former 
method  is  of  course  the  usual  one  in  practice.  It  has  been 
suggested  that  the  bacteria  contained  in  the  rennet  exert  a 
considerable  influence  on  the  cheese,  but  this,  although 
rennet  contains  bacteria,  is  hardly  established.  It  is  not 
here,  however,  that  bacteria  really  play  their  role.  After 
this  physical  separation,  when  the  cheese  is  pressed  and  set 
aside,  is  the  period  for  the  commencement  of  the  ripening 
process. 

That  bacteria  perform  the  major  part  of  this  ripening  pro- 
cess, and  are  essential  to  it,  is  proved  by  the  fact  that  when 
they  are  either  removed  or  opposed  the  curing  changes  im- 
mediately cease.  If  the  milk  be  first  sterilised,  or  if  anti- 
septics, like  thymol,  be  added,  the  results  are  negative.  It 
is  not  yet  known  whether  this  peptonising  process  is  due  to 
the  influence  of  a  single  organism  or  not.  The  probability, 
however,  is  that  it  is  to  be  ascribed  to  the  action  of  that 
group  of  bacteria  known  as  the  lactic-acid  organisms.  Nor 
is  it  yet  known  whether  the  peptonisation  of  the  casein  and 
the  production  of  the  flavour  are  the  results  of  one  or  more 
species.  Freudenreich  believes  them  to  be  due  to  two 
different  forms. 

However  that  may  be,  we  meet  with  at  least  four  com- 
mon groups  of  bacteria  more  or  less  constantly  present  in 


222  BACTERIA 

cheese-ripening,  either  in  the  early  or  late  stages.  First, 
there  are  the  lactic-acid  bacteria,  by  far  the  largest  group, 
and  the  one  common  feature  of  which  is  the  production  by 
fermentation  of  lactic  acid ;  secondly,  there  are  the  casein- 
digesting  bacteria,  present  in  relatively  small  numbers; 
thirdly,  the  gas-producing  bacteria,  which  give  to  cheese  its 
honeycombed  appearance;  lastly,  an  indifferent  or  miscel- 
laneous group  of  extraneous  bacteria,  which  were  in  the  milk 
at  the  outset  of  cheese-making,  or  are  intruders  from  the 
air  or  rennet.  All  these  four  groups  may  bring  about  a 
variety  of  changes,  beneficial  and  otherwise,  in  the  cheese- 
making. 

In  order  that  the  relation  of  bacteria  to  cheese  may  be 
more  fully  understood,  we  may  draw  attention  to  some  ex- 
periments conducted  by  Professor  H.  L.  Russell  as  to  the 
numbers  of  bacteria  present  during  different  stages  of  the 
ripening,  excluding  those  already  referred  to  as  present  in 
the  rennet.  It  appears  that  there  is  always  at  first  a  marked 
increase  in  the  number  of  micro-organisms,  which  is  soon 
followed  by  a  more  gradual  decline.  While  the  casein- 
digesting  and  gas-producing  classes  suffer  a  general  and 
more  or  less  rapid  decline,  the  lactic-acid  bacteria  develop 
to  an  enormous  extent,  from  which  fact  it  would  appear 
that  cheese  offers  ideal  conditions  for  the  development  of 
the  latter.  In  some  most  interesting  records  Professor  Rus- 
sell has  divided  the  ripening  process  into  three  divisions: 

1 .  Period  of  Initial  Bacterial  Decline  in  Cheese.     Wh e  re  the 
green  cheeses  were  examined  immediately  after  removing 
from  the  press,  it  was  usually  found  that  a  diminution  in 
numbers  of  bacteria  had  taken  place.     This  period  of  de- 
cline lasts  but  a  short  time,  not  beyond  the  second  day. 
Lower  temperature  and  expulsion  of  the  whey  would  ac- 
count for  this  general  decline  in  all  species  of  bacteria. 

2.  Period  of  Bacterial  Increase.    Soon  after  the  cheese  is  re- 
moved from  the  press  a  most  noteworthy  change  takes  place 


BACTERIA    IN  FOODS 


223 


in  green  cheese.  A  very  rapid  increase  of  bacteria  occurs, 
confined  almost  exclusively  to  the  lactic-acid  group.  This 
commences  in  green  cheese  about  the  eighth  day,  and  con- 
tinues more  or  less  for  twenty  days.  In  Cheddar  cheese  it 
commences  about  the  fifth  day,  reaches  its  maximum  about 
the  twentieth  day,  declines  rapidly  to  the  thirtieth  day,  and 
gradually  for  a  hundred  following  days.  During  the  first 
forty  days  of  this  period  the  casein-digesting  and  gas-pro- 
ducing organisms  are  present,  and  at  first  increasing,  but 
relatively  to  only  a  very  slight  degree.  With  this  rapid 
increase  in  organisms  the  curd  begins  to  lose  its  elastic 
texture,  and  before  the  maximum  number  of  bacteria  is 
reached  the  curing  is  far  advanced.  Freudenreich  has 
shown  that  acid  inhibits  the  growth  of  the  casein-digesting 
microbes  and  vice  versd. 

3.  Period  of  Final  Bacterial  Decline.  The  cause  of  this 
decline  can  only  be  conjectured,  but  it  is  highly  probable 
that  it  is  due  to  a  general  principle  to  which  reference  has 
frequently  been  made,  viz.,  that  after  a  certain  time  the 
further  growth  of  any  species  of  bacteria  is  prevented  by  its 
own  products.  We  may  observe  that  the  gas-producing 
bacteria  in  Cheddar  cheese  last  much  longer  than  the  pep- 
tonising  organisms,  for  they  are  still  present  up  to  eighty 
days.  Professor  Russell  aptly  compares  the  bacterial  vege- 
tation of  cheese  with  its  anologue  in  a  freshly  seeded  field. 
"At  first  multitudes  of  weeds  appear  with  the  grass.  These 
are  the  casein-digesting  organisms,  while  the  grass  is  com- 
parable to  the  more  native  lactic-acid  flora.  In  course  of 
time,  however,  grass,  which  is  the  natural  covering  of  soil, 
*  drives  out  '  the  weeds,  and  in  cheese  a  similar  condition 
occurs."  In  milk  the  lactic-acid  bacteria  and  peptonising 
organisms  grow  together;  in  ripening  cheese  the  former 
eliminate  the  latter. 

We  have  seen  that  the  conclusion  generally  held  respect- 
ing these  lactic-acid  bacteria  is  that  they  are  the  main  agents 


224  BACTERIA 

in  curing  the  cheese.  Upon  this  basis  a  system  of  pure 
starters  has  been  adopted,  the  characteristics  of  which 
must  be  as  follows :  (a)  The  organism  shall  be  a  pure  lactic- 
acid-producing  germ,  incapable  of  producing  gaseous  pro- 
ducts; (b)  it  should  be  free  from  any  undesirable  aroma;  (c) 
it  should  be  especially  adapted  for  vigorous  development 
in  milk.  The  starter  may  be  propagated  in  pasteurised 
or  sterilised  milk  from  a  pure  culture  from  the  laboratory. 
The  advantages  accruing  from  the  uses  of  this  lactic-acid 
culture,  as  compared  with  cheese  made  without  a  culture, 
are  that  with  sweet  milk  it  saves  time  in  the  process  of 
manufacture;  that  with  tainted  milk,  in  which  acid  develops 
imperfectly,  it  is  an  aid  to  the  development  of  a  proper 
amount  of  acid  for  a  typical  Cheddar  cheese ;  and  that  the 
flavour  and  quality  of  such  cheese  is  preferable  to  cheese 
which  has  not  been  thus  produced.  Professor  Russell  is  of 
opinion  that  the  lactic-acid  organisms  are  to  be  credited 
with  greater  ripening  powers  than  the  casein-digesting 
organisms,  but  it  must  not  be  forgotten  that  these  two  great 
families  of  bacteria  are  still  more  or  less  on  trial,  and  it  is 
not  yet  possible  finally  to  dispose  of  either  of  them.  Mr. 
F.  J.  Lloyd  holds  that  though  "  the  greater  the  number  of 
lactic-acid  bacilli  in  the  milk  the  greater  the  chance  of  a 
good  curd,"  still  "  this  organism  alone  will  not  produce  that 
nutty  flavour  which  is  so  sought  after  as  being  the  essential 
characteristic  of  an  excellent  Cheddar  cheese."  ' 

There  are  several  difficulties  to  be  encountered  by  dairy- 
men starting  a  ripening  by  the  addition  of  a  pure  culture. 
To  begin  with,  there  is  the  initial  difficulty  of  not  being  able 
to  pasteurise  milk  intended  for  cheese,  as  rennet  will  not 
coagulate  pasteurised  milk  (Lloyd).  Hence  it  is  impossible 

1  "  Observations  on  Cheddar  Cheese  Making,"  Reports  of  Bath  and  West 
and  Southern  Counties  Society,  1898,  pp.  163-171.  Mr.  Lloyd's  Reports  to 
the  West  of  England  Society  since  1892  contain  various  points  respecting  the 
application  of  bacteriology  to  cheese-making. 


BACTERIA    IN  FOODS  22$ 

to  avoid  some  contamination  of  the  milk  previous  to  the 
addition  of  the  culture.  The  continual  uncontaminated 
supply  of  pure  culture  is  by  no  means  an  easy  matter.  The 
maintenance  of  a  low  temperature  to  prevent  the  rapid  mul- 
tiplication of  extraneous  bacteria  will,  in  some  localities,  be 
a  serious  difficulty.  These  difficulties  have,  however,  not 
proved  insurmountable,  and  by  various  workers  in  various 
localities  and  countries  culture-ripening  is  being  carried  on. 

Abnormal  Ripening.  Unfortunately,  from  one  cause  or 
another,  faulty  fermentations  and  changes  are  not  infre- 
quently set  up.  Many  of  these  may  be  prevented,  being 
due  to  lack  of  cleanliness  in  the  process  or  in  the  milking; 
others  are  due  to  the  gas-producing  bacteria  being  present 
in  abnormally  large  numbers.  When  this  occurs  we  obtain 
what  is  known  as  "  gassy  "  cheese,  on  account  of  its  sub- 
stance being  split  up  by  innumerable  cavities  and  holes  con- 
taining carbonic  acid  gas,  or  sometimes  ammonia  or  free 
nitrogen.  Some  twenty-five  species  of  micro-organisms 
have  been  shown  by  Adamety  to  cause  this  abnormal  swell- 
ing. In  severe  cases  of  this  gaseous  fermentation  the  pro- 
duct is  rendered  worthless,  and  even  when  less  marked  the 
flavour  and  value  are  much  impaired.  Winter  cheese 
contains  more  of  this  species  of  bacteria  than  summer. 
Acid  and  salt  are  both  used  to  inhibit  the  action  of  these 
gas-producing  bacteria  and  yeasts,  and  with  excellent  results. 

We  may  remark  that  the  character  of  the  gas  holes  in 
cheese  is  not  of  import  in  the  differentiation  of  species.  If 
a  few  gas  bacteria  are  present,  the  holes  will  be  large  and 
less  frequent;  if  many,  the  holes  will  be  small,  but  numer- 
ous. (Swiss  cheese  having  this  characteristic  is  known  as 
Nissler  cheese.) 

Many  of  these  gas  germs  belong  to  the  lactic-acid  group, 
and  are  susceptible  to  heat.  A  temperature  of  140°  F. 
maintained  for  fifteen  minutes  is  fatal  to  most  of  them, 

largely  because  they  do  not  form  spores.     The  sources  of 
15 


226  BACTERIA 

the  extensive  list  of  bacteria  found  in  cheese  are  of  course 
varied,  more  varied  indeed  than  is  the  case  with  milk.  For 
there  are,  in  addition  to  the  organisms  contained  in  the  milk 
brought  to  the  cheese  factory,  the  following  prolific  sources, 
viz.,  the  vats  and  additional  apparatus;  the  rennet  (which 
itself  contains  a  great  number);  the  water  that  is  used  in 
the  manufacture. 

In  addition  to  the  abnormalities  due  to  gas,  there  are  also 
other  faulty  types.  The  following  chromogenic  conditions 
occur:  red  cheese,  due  to  a  micrococcus;  blue  cheese,  pro- 
duced, according  to  Vries,  by  a  bacillus;  and  black  cheese, 
caused  by  a  copious  growth  of  low  fungi.  Bitter  cheese  is 
the  result  of  the  Micrococcus  casei  amari  of  Freudenreich,  a 
closely  allied  form  of  Conn's  micrococcus  of  bitter  milk. 
Sometimes  cheese  undergoes  a  putrefactive  decomposition, 
and  becomes  more  or  less  putrid.  These  latter  conditions, 
like  the  gassy  cheeses,  are  due  to  the  intrusion  of  bacteria 
from  without,  or  from  udder  disease  of  the  cow.  Healthy 
cows,  clean  milking,  and  the  introduction  of  pure  cultures 
are  the  methods  to  be  adopted  for  avoiding  "  diseases  "  of 
cheese  and  obtaining  a  well-flavoured  article  which  will 
keep. 

Finally,  we  may  quote  five  conclusions  from  the  prolonged 
researches  of  Mr.  Lloyd  1  which  cannot  but  prove  helpful  to 
the  Cheddar  cheese  industry  in  England : 

1.  To   make  Cheddar  cheese  of  excellent  quality,    the 
Bacillus  acidi  lactici  alone  is  necessary;  other  germs  will 
tend  to  make  the   work   more   rather   than    less  difficult. 
Hence  scrupulous  cleanliness  should  be  a  primary  consider- 
ation of  the  cheese-maker. 

2.  No  matter  what  system  of  manufacture  be  adopted,  two 
things  are  necessary.     One  is  that  the  whey  be  separated 
from  the  curd,  so  that  when  the  curd  is  ground  it  shall  con- 
tain not  less  than  40  per  cent,  of  water,  and  not  more  than 

1  Journal  of  Bath  and  West  of  England  Society,  1893,  1895,  and  1897. 


BACTERIA   IN  FOODS  22/ 

43  per  cent. ;  the  other  point  is  that  the  whey  left  in  the 
curd  shall  contain,  developed  in  it  before  the  curd  is  put  in 
the  press,  at  least  I  per  cent,  of  lactic  acid  if  the  cheese  is 
required  for  sale  within  four  months,  and  not  less  than  8 
per  cent,  of  lactic  acid  if  the  cheese  is  to  be  kept  ripening  for 
a  longer  period. 

3.  The  quality  of  the  cheeses  will  vary  with  the  quality 
of  the  milk  from  which  they  have  been  made,  and  propor- 
tionately to  the  amount  of  fat  present  in  that  milk. 

4.  "  Spongy  curd  "  is  produced  by  at  least  five  organisms, 
and  one  of  these  is  responsible  for  a  disagreeable  taint  found 
in  curd.     They  occur  in  water.     Hence  the  desirability  of 
securing  clean  water  for  all  manipulative  purposes,  and  also 
for  the  drinking  purposes  of  the  milch  cow. 

5.  The  fact  that  certain  bacteria  are    found  in  certain 
localities  and  dairies  is  due  more  to  local  conditions  than  to 
climatic  causes. 

It  is  needless  to  remark  that  these  conclusions  once  more 
emphasise  the  fact  that  strict  and  continual  cleanliness  is  the 
one  desideratum  for  bacteriologically  good  dairying.  That 
being  secured  in  the  cow  at  the  milking,  in  the  transit,  and 
at  the  dairy,  it  is  a  comparatively  simple  step,  by  means  of 
pasteurisation  and  the  use  of  good  pure  cultures  of  flavour- 
ing bacteria,  to  the  successful  application  of  bacteriology  to 
dairy  produce. 

Methods  of  Examination  of  Milk  : 

i .  Preparation  of  Microscopic  Slides.  This  course  might 
at  once  occur  to  the  mind  as  the  first  to  adopt  in  searching 
for  bacteria  in  milk.  Devices  have  accordingly  been  pro- 
posed for  saponification  previous  to  staining.  Some  recom- 
mend the  addition  of  a  few  drops  of  a  solution  of  sodium 
carbonate;  others  use  methylene  blue  and  chloroform. 
But,  whatever  plan  of  staining  is  adopted,  this  method  of 
examination  in  its  simplest  form  is  in  no  degree  a  criterion 
of  the  bacterial  content  of  a  large  quantity  of  milk. 


228 


BACTERIA 


Hence  it  has  come  to  be  recognised  that  one  of  two 
manipulations  must  precede  such  microscopic  examination. 
These  simple  processes  are  known  by  the  terms  of  sediment- 
ation and  centrifugalisation.  Sedimentation  means  merely 
placing  the  milk  in  conical  glasses  in  a  cool 
place  for  twenty-four  hours.  The  intro- 
duction of  improved  forms  of  the  centri- 
fuge has  brought  the  second  method  of 
securing  a  sediment  into  preference.  Five 
cubic  centimetres  of  the  milk  are  intro- 
duced into  the  graduated  bottle,  which  is 
then  placed  in  the  centrifuge,  and  whirled 
for  one  or  two  minutes.  Thus  a  deposit 
of  particulate  matter  is  ensured.  Cover- 
glass  specimens  of  the  sediment  or  deposit 
are  then  prepared  and  stained  in  the  ordi- 
nary way. 

In  testing  for  tubercle  something  more 
is  generally  necessary.  To  the  50  cc.  of 
the  milk  set  aside  for  sedimentation  10  cc. 
of  liquefied,  colourless  carbolic  acid  are 
added.  The  mixture  is  shaken  and  poured 
into  the  conical  glass.  After  standing  for 
twenty-four  hours  a  little  of  the  sediment 
is  taken  by  means  of  a  pipette  and  exam- 
ined by  ordinary  methods,  though  after 
"  fixing  "  the  films  with  heat  they  are 
some  times  passed  through  equal  parts  of 
alcohol  and  ether.  The  stain  is  of  course 
that  usually  adopted  in  tubercle,  namely,  the  Ziehl-Neelsen. 
Scheurlen  suggested  a  method  for  demonstrating  the  tu- 
bercle bacillus  in  milk  by  steeping  the  cover  glasses  first  in 
alcohol  and  then  ether,  after  which  they  were  stained  with 
Ziehl-Neelsen. 

2.  Plate  Culture.     The  milk  is  to  be  diluted  a  thousand  or 


A  CENTRIFUGE 

Used  in  the  Examination 
of  Milk 


BACTERIA   IN  FOODS  2 29 

more  times  with  sterile  water,  and  ordinary  plate  cultures 
made  in  Petri  dishes  or  flat-bottomed  conical  flasks.  The 
colonies  should  be  counted  as  late  as  possible;  but  even 
then  the  isolation  of  pathogenic  germs  is  uncertain.  As 
regards  further  procedure,  the  ordinary  methods  of  sub- 
culturing  adopted  in  water  examination  must  be  strictly 
followed,  and  the  special  tests  for  Bacillus  typhosus  and  B. 
coli  applied.  As  we  have  already  seen,  the  quantitative 
estimation  of  organisms  in  milk  is  not  of  the  same  value  as 
in  water. 

3.  Inoculation.  To  test  the  capacity  of  the  milk  for  caus- 
ing disease,  before  or  after  centrifugalisation,  preferably  the 
latter,  a  certain  quantity  of  the  sediment  may  be  inoculated 
into  guinea-pigs.  In  suspected  tubercle  2  cc.  may  be  taken  ; 
in  diphtheria  a  little  less  will  suffice.  The  inoculation 
should  be  either  intraperitoneal  or  subcutaneous.  Many 
authorities  hold  that  this  test  is  the  only  safe  one  to  protect 
the  public  from  milk  containing  germs  of  disease. 

BACTERIA   IN   OTHER   FOODS 

Shell-fish  have  recently  claimed  the  attention  of  bacterio- 
logists, owing  to  the  outbreak  of  typhoid  and  other  epidemics 
apparently  traceable  to  oysters. 

It  is  four  or  five  years  since  Professor  Conn  startled  the 
medical  world  by  tracing  an  epidemic  of  typhoid  fever  to 
the  consumption  of  some  uncooked  oysters.1  Almost  at  the 
same  time  Sir  William  Broadbent  published  in  the  British 
Medical  Journal  a  series  of  cases  occurring  in  his  practice 
which  illustrated  the  same  channel  of  infection.  Since  then 
a  number  of  similar  items  of  evidence  to  the  same  effect 
have  cropped  up.  Hence  there  is  little  wonder  that  a  num- 
ber of  investigators  concentrated  their  attention  upon  this 
matter.  Professors  Herdman  and  Boyce,  of  Liverpool,  Dr. 
Cartwright  Wood,  Dr.  Klein,  and  Dr.  Timbrell  Bulstrode 

1  New  York  Medical  Record,  1894. 


230  BACTERIA 

are  some  of  the  chief  contributors  to  the  elucidation  of  this 
problem. 

The  mode  of  infection  of  oysters  by  pathogenic  bacteria 
is  briefly  as  follows :  The  sewage  of  certain  coast  towns  is 
passed  untreated  out  to  sea.  At  or  near  the  outfall,  oyster- 
beds  are  laid  down  for  the  purpose  of  fattening  oysters. 
Thus  they  become  contaminated  with  saprophytic  and  path- 
ogenic germs  contained  in  the  sewage.  It  will  be  at  once 
apparent  that  several  preliminary  questions  require  attention 
before  any  deductions  can  be  drawn  as  to  whether  or  not 
oysters  convey  virulent  disease  to  consumers.  To  the  solu- 
tion of  these  Dr.  Cartwright  Wood  was  one  of  the  first  to 
address  himself. 

The  precise  conditions  which  render  one  locality  more 
favourable  than  another  in  respect  to  oyster  culture  are  not 
fully  known.  But  it  has  been  observed  that  they  do  not 
flourish  in  water  containing  less  than  three  per  cent,  of  salt. 
Hence  they  are  absent  from  the  Baltic  Sea,  which,  owing  to 
the  fresh  water  flowing  into  it  in  rivers,  contains  a  smaller 
percentage  of  salt  than  three.  Oysters  appear  in  addition, 
to  favour  a  locality  where  they  find  their  chosen  food  of 
small  animalculae  and  particles  of  organic  matter.  Such  a 
favourable  locality  is  the  mouth  of  a  river,  where  tides  and 
currents  also  assist  in  bringing  food  to  the  oyster.  Unfor- 
tunately, however,  in  a  crowded  country  like  England  such 
localities  round  her  coasts  are  frequently  contaminated  by 
sewage  from  outfalls.  Thus  the  oysters  and  the  sewage 
come  into  intimate  relation  with  each  other. 

Professor  Giaxa  carried  out  some  experiments  in  1889  at 
Naples  which  appeared  to  show  that  the  bacilli  of  cholera 
and  typhoid  rapidly  disappeared  in  ordinary  sea-water. 
Other  observers  at  about  the  same  time,  notably  Foster  and 
Freitag,  arrived  at  an  opposite  conclusion.  In  1894  Profes- 
sor Percy  Frankland,  in  a  report  to  the  Royal  Society,  de- 
clared "  that  common  salt,  whilst  enormously  stimulating 


BACTERIA   IN  FOODS  231 

the  multiplication  of  many  forms  of  water  bacteria,  exerts  a 
directly  and  highly  prejudicial  effect  on  the  typhoid  bacilli, 
causing  their  rapid  disappearance  from  the  water,  whether 
water  bacteria  are  present  or  not."  It  was  at  this  time, 
when  the  matter  was  admittedly  in  an  unsatisfactory  stage, 
that  Dr.  Cartwright  Wood  made  his  experiments.1  We 
have  not  space  here  to  enter  into  this  work.  But  his  con- 
clusions seem  to  have  been  amply  established,  and  were  to 
the  effect  that  typhoid  and  cholera  bacilli  could,  as  a  matter 
of  fact,  exist  over  very  lengthened  periods  in  ordinary  sea- 
water.  The  next  step  was  to  demonstrate  the  length  of 
time  the  bacilli  of  cholera  remained  alive  in  the  pallial  cavity 
and  body  of  the  oyster.  Dr.  Wood  found  they  did  so  for 
eighteen  days  after  infection,  though  in  greatly  diminished 
numbers.  This  diminution  was  due  to  one  or  all  of  three 
reasons :  (a)  the  effect  of  the  sea-water  already  referred  to  as 
finally  prejudicial  to  bacilli  of  typhoid;  (b)  the  vital  action 
of  the  body-cells  of  the  oyster;  (c)  the  washing  away  of 
bacilli  by  the  water  circulating  through  the  pallial  cavity. 

It  will  have  been  noticed  that  up  to  the  present  we  have 
learned  that  typhoid  bacilli  can  and  do  live  in  sea-water, 
and  also  inside  oysters  up  to  eighteen  days,  but  in  ever- 
diminishing  quantities.  The  question  now  arises:  What  is 
the  influence  of  the  oyster  upon  the  contained  bacilli  ? 
Under  certain  conditions  of  temperature  organisms  may 
multiply  with  great  rapidity  inside  the  shell  of  the  oyster. 
Yet,  on  the  other  hand,  the  amoeboid  cells  of  the  oyster, 
the  acid  secretion  of  its  digestive  glands,  or  the  water  circu- 
lating through  its  pallial  cavity,  may  act  inimically  on  the 
germs.  Proof  can  be  produced  in  favour  of  the  third  and 
last-named  mode  by  which  an  oyster  can  cleanse  itself  of 
germs.  So  far,  then,  we  have  met  with  no  facts  which 
make  it  impossible  for  oysters  to  contain  for  a  lengthened 
period  the  specific  bacteria  of  disease.  Let  us  now  turn  to 

1  British  Medical  Journal,  1896,  ii.,  p.  760  et  seq. 


232  BACTERIA 

their  opportunity  for  acquiring  such  disease  germs.  It  is 
afforded  them  during  the  process  of  what  is  termed  "  fatten- 
ing."  By  this  process  the  body  of  the  oyster  acquires  a 
plumpness  and  weight  which  enhances  its  commercial  value. 
This  desired  condition  is  obtained  by  growing  the  oyster  in 
'  brackish  "  water,  for  thus  it  becomes  filled  out  and  me- 
chanically distended  with  water.  But  if  this  water  contains 
germs  of  disease,  what  better  opportunity  could  such  germs 
have  for  multiplication  than  within  the  body-cavity  of  an 
oyster  ?  "  The  contamination  of  sea-water,  therefore,  in 
the  neighbourhood  of  oyster-beds  may  undoubtedly  lead  to 
the  molluscs  becoming  infected  with  pathogenic  organisms  " 
(Wood).  Yet  we  have  seen  that,  apart  altogether  from  the 
individual  susceptibilites  or  otherwise  of  the  consumer,  there 
are  in  the  series  of  events  necessary  to  infection  many  occa- 
sions when  circumstances  would  practically  free  the  oysters 
from  infection. 

The  sources  of  pollution  of  oysters  are  not  the  fattening 
beds  alone.  The  native  beds  also  may  afford  opportunity 
for  contamination.  Thirdly,  in  packing  and  transit,  and 
fourthly,  in  storage  in  shops  and  warehouses,  there  is  fre- 
quently abundant  facility  for  putrefactive  bacteria  to  gain 
entrance  to  the  shells  of  oysters. 

Dr.  Klein's  researches  *  into  this  question  have  been 
wholly  confirmatory  of  the  facts  elicited  by  Dr.  Cartwright 
Wood.  Despite  the  tendency  of  the  bacilli  of  cholera  and 
typhoid  to  die  out  quickly  in  crude  sewage,  the  sewage  is 
sufficiently  altered  or  diluted  at  the  outfall  for  these  organ- 
isms to  exist  there  in  a  virulent  state.  We  may  give  Dr. 
Klein's  conclusions: 

1.  That  the  cholera  and  typhoid  bacilli  are  difficult  of 
demonstration  in  sewage  known  to  have  received  them. 

2.  Both  organisms  may  persist  in  sea-water  tanks  for  two 

1  Special  Report  of  the  Medical  Officer  to  the  Local  Government  Board  on 
Oyster  Culture,  etc.,  1896. 


BACTERIA    IN  FOODS  233 

or  three  weeks,  the  typhoid  bacillus  retaining  its  character- 
istics unimpaired,  the  cholera  bacillus  tending  to  lose  them. 

3.  Oysters  from    sources    free    of   sewage   contained    no 
bacteria  of  sewage. 

4.  Oysters  from  sources  exposed  to  risk  of  sewage  con- 
tamination   did    contain    colon    bacilli    and    other    sewage 
bacteria. 

5.  In  one  case  Eberth's  typhoid  bacillus  was  found  in  the 
mingled  body  and  liquor  of  the  oyster. 

Nor  do  typhoid  bacilli  lose  activity  or  virulence  by  passing 
through  an  oyster. 

These  researches  once  and  for  all  established  the  fact  that 
oysters  ordinarily  grown  on  oyster-beds  contaminated  with 
bacteria  may,  and  do  on  occasion,  contain  the  virulent 
specific  bacillus  of  typhoid,  which  can  live  both  in  sea-water 
and  within  the  shell  of  the  oyster.  This  being  so,  it  will 
probably  appear  to  the  reader  that  the  risk  of  infection  of 
typhoid  by  oysters  is  very  serious  indeed.  Yet  in  actual 
practice  many  conditions  have  to  be  fulfilled.  For,  in  ad- 
dition to  the  fact  that  the  oysters  must  be  consumed,  as 
is  usual,  uncooked,  the  following  conditions  must  also  be 
present : 

(a)  Each  infective  oyster  must  contain  infected  sewage, 
which  presupposes  that  typhoid  excreta  from  patients  suffer- 
ing from  the  disease  have  passed  into  that  particular  sewage 
untreated  and  not  disinfected. 

(U)  The  infective  oyster  must  be  fed  upon  infected  sew- 
age, and  still  contain  the  virus  in  its  substance. 

(c)  It  has  to  be  eaten  by  a  susceptible  person. 

(d)  There  must  have  been  no  period  of  natural  cleansing 
after  "  fattening." 

Even  to  this  formidable  list  of  conditions  we  must  add  the 
further  remark  that,  owing  to  the  vitality  of  the  body-cells 
of  the  oyster,  or  to  the  lessened  vitality  of  the  bacilli  of 
cholera  and  typhoid,  it  is  generally  the  case  that  the  tend- 


234  BACTERIA 

ency  of  these  organisms  is  rather  to  decrease  and  die  out 
than  live  and  multiply. 

We  shall  probably  maintain  a  satisfactory  balance  of  truth 
if  we  place  alongside  these  facts  the  summary  of  the  Local 
Government  Board  Report. 

"  There  can  be  no  doubt,"  wrote  Sir  Richard  Thorne,  "  that 
oysters  which  have  been  brought  into  sustained  relation  with  the 
typhoid  bacillus  are  liable  to  exhibit  that  microbe  within  the  shell 
contents  and  to  retain  it  for  a  while  under  circumstances  not  only 
permitting  its  rapid  multiplication  when  transferred  again  to  ap- 
propriate media,  but  conserving  at  the  same  time  its  ability  to 
manifest  its  hurtful  properties." 

From  what  has  been  said  the  preventive  treatment  is 
obvious.  All  oyster-layings  and  shell-fish  beds  round  the 
coast  should  be  superintended  and  inspected  by  the  sanitary 
authority  of  the  Government.  The  importation  of  foreign 
oysters,  grown  on  uncontrolled  beds,  should,  if  possible,  be 
restricted  or  supervised.  Further,  as  a  protective  measure  of 
the  first  importance,  oysters  should  be  cleansed,  after  fat- 
tening on  a  contaminated  bed,  by  being  deposited  for  several 
weeks  at  some  point  along  the  coast  which  is  washed  by 
pure  sea-water.  Retention  in  dirty  water-tanks,  in  uncleanly 
shops  and  warehouses,  is  also  to  be  greatly  deprecated. 

In  order  to  examine  oysters  bacteriologically,  it  is  neces- 
sary to  pay  particular  attention  to  the  water  in  the  pallial 
cavity,  the  contents  of  the  alimentary  canal,  and  the  wash- 
ings of  the  shell  itself.  Ordinary  media  may  be  used  for 
obtaining  a  growth  of  the  contained  organisms. 

Other  shell-fish  than  oysters  do,  from  time  to  time,  cause 
epidemics  or  individual  cases  of  gastro-intestinal  irritation, 
and  probably  contain  various  germs.  These  they  acquire  in 
all  probability  from  their  food,  which  by  their  own  choice 
is  frequently  of  a  doubtful  character. 

Meat.  Parasites  are  occasionally  found  in  meat,  but 
bacteria  are  comparatively  rare.  Not  that  they  do  not 


BACTERIA   IN  FOODS  23$ 

occur  in  the  bodies  of  animals  used  for  human  consumption, 
for  in  the  glands,  mesenteries,  and  other  organs  they  are 
common.  But  in  those  portions  of  the  carcass  which  are 
used  by  man,  namely  the  muscles,  bacteria  are  rare.  The 
reasons  alleged  for  this  are  the  acid  reaction  (sarcolactic 
acid)  and  the  more  or  less  constant  movement  during  life. 
A  bacterial  disease  which,  perhaps  more  than  any  other, 
might  be  expected  to  be  conveyed  by  meat  is  tubercle.  Yet 
the  recent  Royal  Commission  on  Tuberculosis  has  again 
emphasised  the  absence  of  bacilli  in  the  meat  substance: 

"  In  tissues  which  go  to  form  the  butcher's  joint,  the  material 
of  tubercle  is  not  often  found  even  where  the  organs  (lungs,  liver, 
spleen,  membranes,  etc.)  exhibit  very  advanced  or  generalised 
tuberculosis;  indeed,  in  muscle  and  muscle  juice  it  is  very  seldom 
that  tubercle  bacilli  are  to  be  met  with ;  perhaps  they  are  some- 
what more  often  to  be  discovered  in  bone,  or  in  some  small 
lymphatic  gland  embedded  in  intermuscular  fat."  1 

The  only  way  in  which  such  meat  substance  becomes  in- 
fected with  tubercle  appears  to  be  through  carelessness  in 
the  butcher,  who  perchance  smears  the  meat  substance  with 
a  knife  that  has  been  used  in  cutting  the  organs,  and  so  has 
become  contaminated  with  infected  material.  Very  instruct- 
ive also  are  the  results  at  which  Dr.  Sims  Woodhead  arrived 
in  compiling  evidence  for  the  same  Commission  on  the  effect 
of  cooking  upon  tuberculous  meat : 

"  Ordinary  cooking,  such  as  boiling  and  more  especially  roast- 
ing, though  quite  sufficient  to  sterilise  the  surface,  and  even  the 
substance  for  a  short  distance  from  the  surface  of  a  joint,  cannot 
be  relied  upon  to  sterilise  tubercular  material  included  in  the 
centre  of  rolls  of  meat,  especially  when  these  are  more  than  three 
pounds  or  four  pounds  weight.  The  least  reliable  method  of 
cooking  for  this  purpose  is  roasting  before  a  fire;  next  comes 
roasting  in  an  oven,  and  then  boiling."  a 

1  Royal  Commission  on  Tuberculosis,  Report,  1895,  pt.  i.,  p.  13. 
*Ibid.,  p.  18. 


236  BACTERIA 

From  this  statement  it  will  be  understood  that  rolled  meat 
may  be  a  source  of  infection  to  a  greater  degree  than  the 
ordinary  joint. 

Notwithstanding  this  negative  evidence,  more  than  twenty 
species  of  bacteria  have  been  isolated  from  canned  meats 
and  hams,  and  a  considerable  number  of  poisoning  cases 
have  occurred  from  meat  contaminated  with  bacteria  or  their 
products.  The  general  symptoms  of  such  meat  poisoning 
are  vomiting,  diarrhoea,  fever,  and  more  or  less  prostration. 
Ballard  and  Klein  isolated  a  specific  microbe  from  samples 
of  bacon  which  appear  to  have  caused  an  epidemic  of  in- 
fectious pneumonia  at  Middlesborough.  In  1880  occurred 
the  well-known  "  Welbeck  disease  "  epidemic.  A  public 
luncheon  was  followed  by  severe  and  even  fatal  illness. 
Seventy-two  persons  were  affected,  and  four  died.  A  spe- 
cific bacillus  was  isolated  by  Klein.  In  1881  much  the 
same  thing  happened  at  Nottingham,  in  which  fifteen  per- 
sons were  attacked,  and  one  died.  The  same  bacillus  was 
isolated  from  the  pernicious  pork.  Again  in  1889  an  out- 
break of  diarrhoea  at  Carlisle  was  traced  to  bacterially  dis- 
eased pork.  But  taking  these  and  similar  cases  at  their 
worst,  there  can  be  no  doubt  that  under  no  circumstances  is 
meat  as  infective  as  milk. 

Ice-cream.  In  1894  Dr.  Klein  had  occasion  to  bacterio- 
logically  examine  ice-creams  sold  in  the  streets  of  London. 
In  all  six  samples  were  analysed,  and  in  each  sample  the 
conclusions  resulting  were  of  a  nature  sufficiently  serious  to 
support  the  view  that  the  bacterial  flora  was  not  inferior  to 
ordinary  sewage.  The  water  in  which  the  ice-cream  glasses 
were  washed  was  also  examined,  and  found  to  contain  large 
numbers  of  bacteria. 

Since  that  date  many  investigations  have  been  made  into 
ice-creams.  It  appears  that  they  are  often  made  under  ex- 
tremely foul  circumstances,  and  with  anything  but  sterilised 
appliances.  Little  wonder,  then,  that  the  numbers  of  bac- 


BACTERIA    IN  FOODS 


237 


teria  present  run  into  millions.  In  nearly  all  recorded  cases 
the  quality  of  the  germs  as  well  as  the  quantity  has  been  of 
a  nature  to  cause  some  concern.  Bacillus  coli  communis, 
which,  though  not  now  considered  absolutely  indicative  of 
alimentary  pollution,  is  looked  upon  as  a  highly  unsatisfac- 
tory inhabitant  of  water,  has  been  found  in  considerable 
abundance.  The  Proteus  family,  which  also  possesses  a 
putrefactive  function,  is  common  in  ice-creams.  The  com- 
mon water  bacteria  are  nearly  always  present. 

Bacillus  typhosus  itself,  it  is  said,  has  been  isolated  from 
some  ice-cream  which  was  held  responsible  for  an  outbreak 
of  enteric  fever.  The  material  had  become  infected  during 
process  of  manufacture  in  the  house  of  a  person  suffering 
from  unnotified  typhoid  fever. 

Now,  whilst  reports  of  the  above  nature  appear  very 
alarming,  the  fact  is  that  hundreds  of  weakly  children  de- 
vour ice-cream  with  apparent  impunity,  and  when  evil  fol- 
lows it  is  not  infrequently  due  to  other  than  bacterial 
conditions.  The  cold  mass  itself  may  inhibit  the  resistance 
of  the  gastric  tissues.  Tyrotoxicon,  the  alkaloid  separated 
from  cheese  and  cream  by  Vaughan,  may  be  responsible  for 
some  alimentary  irritation.  On  the  whole,  the  practical 
effect  upon  the  community  is  not  in  proportion  to  the 
bacterial  content  of  the  ice-cream.  Yet,  nevertheless,  we 
ought  to  be  much  more  watchful  than  in  the  past  to  preserve 
ice-cream  from  pollution  with  harmful  bacteria. 

The  two  chief  constituents  which  contribute  their  quota 
of  germ  life  to  ice-cream  are  ice  and  cream.  In  addition, 
the  uncleanly  methods  of  manufacture  render  the  material 
likely  to  contain  the  six  or  seven  millions  of  micro-organisms 
per  cc.  which  have  been  on  several  occasions  estimated. 
To  cleanly  methods  of  dairying  we  have  already  fully  re- 
ferred ;  to  the  bacterial  content  of  milk  and  cream  we  have 
also  paid  some  attention ;  but  we  have  not  had  an  oppor- 
tunity of  saying  anything  of  germs  in  ice. 


238  BACTERIA 

Ice  contains  bacteria  in  varying  quantities  from  20  per  cc. 
to  10,000  or  more.  Nor  is  variation  in  number  affected 
alone  by  the  condition  of  the  water,  for  samples  collected 
from  one  and  the  same  place  differ  widely.  The  quality 
follows  in  large  measure  the  standard  of  the  water. 

Water  bacteria,  Bacillus  coli,  putrefactive  bacteria,  and 
even  pathogenic  have  been  found  in  ice.  Many  of  the 
latter  can  live  without  much  difficulty  and  are  most  numer- 
ous in  ice  containing  air-bubbles. 

Dr.  Prudden,  of  New  York,  performed  a  series  of  experi- 
ments in  1887  to  show  the  relative  behaviour  of  bacteria  in 
ice.  Taking  half  a  dozen  species,  he  inoculated  sterilised 
water  and  reduced  it  to  a  very  low  temperature  for  a  hun- 
dred and  three  days,  with  the  following  results  -.—Bacillus 
prodigiosus  diminished  from  6300  per  cc.  to  3000  within  the 
first  four  days,  to  22  in  thirty-seven  days,  and  vanished 
altogether  in  fifty-one  days;  a  liquefying  water  bacillus, 
numbering  800,000  per  cc.  at  the  commencement,  had  dis- 
appeared in  four  days ;  Stapkylococcus  pyogenes  aureus  and 
B.  fluorescent  showed  large  numbers  present  at  the  end  of 
sixty-six  and  seventy-seven  days  respectively;  B.  typhosus, 
which  was  present  1,000,000  per  cc.  after  eleven  days,  fell 
to  72000  after  77  days,  and  7,000  at  the  end  of  103  days. 
Anthrax  bacilli  are  susceptible  to  freezing,  but  their  spores 
are  practically  unaffected  (Frankland). 

From  these  facts  it  will  be  seen  that  bacteria  live,  but  do 
not  multiply,  in  ice. 

In  making  a  bacterial  investigation  into  the  flora  of  ice- 
cream, it  is  necessary  to  remember  that  considerable  dilution 
with  sterilised  water  is  required.  The  usual  methods  of 
examining  water  and  milk  are  adopted. 

Bread  forms  an  excellent  medium  for  moulds,  but  unless 
specially  exposed  the  bacteria  in  it  are  few.  Waldo  and 
Walsh  have,  however,  demonstrated  that  baking  does  not 
sterilise  the  interior  of  bread.  These  observers  cultivated 


BACTERIA   IN  FOODS  239 

numerous  bacteria  from  the  centre  of  newly  baked  London 
loaves.1  The  writer  has  recently  made  a  series  of  examina- 
tions of  the  air  of  several  underground  bakehouses  in  Cen- 
tral London ;  but,  though  the  air  was  highly  impregnated 
with  flour-dust,  few  bacteria  were  present. 

Other  foods  and  beverages  may  be,  and  are,  from  time  to 
time  contaminated  in  some  small  degree  with  bacteria  or 
their  spores.  Such  contaminations  are  generally  due  to 
uncleanly  manufacture  or  unprotected  storage.  The  prin- 
ciples of  examination  or  of  the  prevention  of  pollution  are 
similar  to  those  already  described. 

1  British  Medical  Journal,  1895,  vol.  ii.,  p.  513. 


CHAPTER  VII 

THE  QUESTION  OF  IMMUNITY  AND  ANTI- 
TOXINS 

THE  term  natural  immunity  is  used  to  denote  natural 
resistance  to  some  particular  specific  disease.  It  may 
refer  to  race,  or  age,  or  individual  idiosyncrasies.  We  not 
infrequently  meet  with  examples  of  this  freedom  from  dis- 
ease. Certain  races  of  men  do  not,  as  a  rule,  take  certain 
diseases.  For  example,  plague  and  leprosy,  though  en- 
demic in  some  countries,  fail  to  get  a  footing  in  England. 
This,,  of  course,  is  due  in  great  measure  to  the  sanitary 
organisation  and  cleanly  customs  of  the  English  people. 
Still,  it  is  also  due  to  the  fact  that  the  English  appear  in 
some  degree  to  be  immune.  Some  authorities  hold  that  the 
immunity  against  leprosy  is  due  to  the  fact  that  the  disease 
has  exhausted  itself  in  the  English  race.  However  that  may 
be,  we  know  that  immunity,  entire  or  partial,  exists.  Child- 
ren, again,  are  susceptible  to  certain  diseases  and  insuscept- 
ible to  certain  others  to  which  older  people  are  susceptible. 
We  know,  too,  that  some  individuals  have  a  marked  pro- 
tection against  some  diseases.  Some  people  coming  into 
the  way  of  infection  at  once  fall  victims  to  the  disease,  whilst 
others  appear  to  be  proof  against  it.  It  is  only  in  recent 
times  that  any  very  intelligent  explanations  have  been 
offered  to  account  for  this  phenomenon.  The  most  recent 
of  these,  and  that  which  appears  to  have  most  to  substanti- 
ate it,  is  known  as  immunity  due  to  antitoxins. 

240 


IMMUNITY  AND  ANTITOXINS  241 

Tke  products  of  bacteria  are  chiefly  six : 

1.  Pigment.     We  have  already  seen  how  many  organisms 
exhibit  their  energy  in  the  formation  of  many  coloured  pig- 
ments.   They  are,  as  a  rule,  "  innocent  "  microbes.    Oxygen 
is  required  for  some,  darkness  for  others,  and  they  all  vary 
according  to  the  medium  upon  which  they  are  growing. 
Red  milk,  yellow  milk,  and  green  pus  afford  examples  of 
pigment  produced  by  bacteria. 

2.  Gas.     Quite  a  number  of  the  common  bacteria,  like 
Bacillus  coli,  produce  gas  in  their  growth ;  hydrogen  (H), 
carbonic  acid  (CO2),    methane  (CH4),   and  even  nitrogen 
(N)  being  formed  by  different  bacteria.     Many  gases  pro- 
duced  during   fermentative  processes   are  the  result,    not 
directly  of  the  growth  of  the  bacillus  causing  the  ferment- 
ation, but  indirectly  owing  to  the  splitting  up  of  the  fer- 
menting fluids. 

3.  Acids.     Lactic,  acetic,  butyric,  etc.,  are  common  types 
of  acids  resulting  from  the  growth  of  bacteria. 

4.  Liquefying  Ferment.     As  we  have  seen,  bacteria  may 
be   classified  with  regard   to   their   behaviour  in  gelatine 
medium,  whether  or  not  they  produce  a  peptonising  ferment 
which  liquefies  the  gelatine. 

5.  Phosphorescence.     Some  species  of  bacteria  in  sea-water 
possess  the  power  of  producing  light. 

6.  Organic  Chemical  Products.     When  a  pathogenic  bacil- 
lus grows  either  in  the  body  or  in  a  test-tube,  it  produces  as 
a  result  of  its  metabolism  certain  poisonous  substances  called 
toxins.     These  may  occur  in  the  blood  as  a  direct  result  of 
the  life  of  the  bacillus,  or  they  may  occur  as  the  result  of  a 
ferment    produced  by   the   bacillus.     They  are  of  various 
kinds  according  to  the  various  diseases,  and  by  their  effect 
upon  the  blood  and  body  tissues  they  cause  the  symptoms 
of  the  disease  in  question.     We  know,  for  instance,  that  a 
characteristic  symptom  common  to  many  diseases  is  fever. 
Now,  fever  is  produced  by  the  action   of  the  albumoses 

16 


242  BACTERIA 

(bodies  allied  to  the  proteids)  upon  the  heat-regulating 
centres  in  the  brain.  Whenever  we  get  a  bacillus  growing 
in  the  body  which  has  the  power  of  producing  a  toxin 
alburnose,  we  get  fever  as  a  result  of  that  product  acting 
upon  the  brain.  Albumoses,  as  a  matter  of  fact,  cause  a 
number  of  symptoms  and  poisonous  effects,  but  the  mention 
of  one  as  an  illustration  will  suffice.  Toxins  act,  roughly 
speaking,  in  two  ways : 

(1)  They  have  a  local  action,  as,  for  example,  in  the  form- 
ation of  an  abscess.     The  presence  of  the  causal  bacteria  in 
the  tissue  brings  about  very  marked  changes.     There  is  a 
multiplication  of  connective-tissue  corpuscles,  an  emigration 
of  leucocytic  cells,  a  congestion  of  blood  corpuscles.     All 
these  elements  assist  in  creating  a  swelling  and  redness,  and 
pain  by  the  subsequent  pressure  upon  the  delicate  nerve 
endings.     These,  as  we  all  know,  are  the  symptoms  of  a 
"  gathering  "  or  abscess.     It  is  a  "  gathering      in  a  strict 
pathological  sense — a  gathering  of  cells  to  oust  the  intruder 
or  build  around  it  a  wall  or  capsule  as  a  protective  measure. 
Now  the  toxin  will  commence  its  local  action.     The  oldest 
cells  in  the  mass  of  congestion  will  be  caused  to  break  down 
into  liquid ;  what  is  called  a  necrosis,  or  death,  will  rapidly 
set  in ;  and  we  shall  have  the  connective-tissue  cells,  leuco- 
cytes, blood  corpuscles,  etc.,  losing  their  form  and  function, 
and  "  coming  to  a  point  "  as  matter,  or  pus.     The  local 
breaking  down  of  these  gatherings  of  cells  into  fluid  matter 
is  believed  to  be  the  work,  not  of  the  bacteria  themselves, 
but  of  their  toxins. 

(2)  Toxins  may  be  absorbed   and   distributed   generally 
throughout  the  body.     They  produce  degenerative  changes 
in  muscles,  in  organs,  and  in  the  blood  itself.     Let  us  take 
diphtheria  as  an  example.     The  bacillus  occurs  in  a  false 
membrane  in  the  throat  and  occasionally  other  parts.     It 
causes  first  the  inflammatory  condition  giving  rise  to  the 
membrane,  and  then  it  breaks  it  down.     In  the  body  of  the 


IMMUNITY  AND  ANTITOXINS  243 

membrane  the  bacillus  appears  to  secrete  a  ferment  which 
by  its  action  and  interaction  with  the  body  cells  and  pro- 
teids,  chiefly  those  of  the  spleen,  produces  albumoses  and  an 
organic  acid.  These  latter  bodies  are  the  toxins.  They 
are  absorbed,  and  pass  throughout  the  body.  There  are 
albumoses,  therefore  we  get  the  frequent  pulse  and  high 
temperature  of  fever;  the  toxins  irritate  the  mucous  mem- 
brane of  the  intestine,  and  cause  various  fermentative 
changes  in  the  contents  of  the  intestine,  therefore  we  get 
the  symptoms  of  diarrhoea;  they  penetrate  the  liver,  spleen, 
and  kidney,  therefore  we  get  fatty  degeneration  and  its  re- 
sults in  these  organs;  they  finally  affect  many  of  the  motor 
and  sensory  nerves,  breaking  up  their  axis  cylinders  into 
globules,  and  therefore  we  get  the  characteristic  paralysis. 
Loss  of  weight  naturally  follows  many  of  these  degenerative 
or  wasting  changes.  Here,  then,  we  have  some  of  the  chief 
changes  set  up  by  the  toxins,  and  these  changes  consti- 
tute the  leading  symptoms  in  the  disease  as  it  is  known 
clinically. 

In  addition  to  the  presence  of  the  specific  bacillus  in  the 
membrane,  we  also  have  a  number  of  other  organisms,  like 
the  Bacillus  coli,  Coccus  Brissou,  Streptococcus  pyogenes,  and 
various  staphylococci,  diplococci,  etc.  Each  of  these  pro- 
duces or  endeavours  in  the  midst  of  keen  competition  and 
strife  to  produce,  its  own  specific  effect.  Thus  we  obtain 
the  complications  of  diphtheria,  for  example  various  sup- 
purative  and  septic  conditions.  The  whole  of  this  com- 
pound process  we  may  tabulate  roughly  as  follows  ' : 

1  It  should  be  distinctly  understood  that  this  table  is  merely  schematic  and 
provisional.  The  details  of  toxin  production  and  its  effect  are  still  open  to 
revision  and  amendment. 


244 


BACTERIA 


Bacillus  coli. 
Coccus  Brissou. 
Staphylococci. 
Diplococci. 
Streptococci. 


Toxins. 


Suppurative  glands, 
septic  poisoning, 
etc. 


BACILLUS  OF  DIPHTHERIA  =  primary  infective 

|  agent. 

Inflammatory  changes  and  fibrinous  exudation. 

FERMENT  IN  MEMBRANE  =  secondary  infective 

|  agent. 

Passes  through  body,  and 

by  digestion  of  proteids 

produces  .         .        \   ALBUMOSES  ; 

\    AN     ORGANIC    ACID. 


1.  Fever. 

2.  Diarrhoea. 

3.  Loss  of  body  weight. 

4.  Fatty  degeneration. 

5.  Degeneration   of   peri- 

pheral   nerves,    and 
resulting  paralysis. 

Such  is  the  general  effect  of  toxins  in  diphtheria.  The 
same  principles  apply  with  equal  force  in  tetanus,  typhoid, 
etc.,  the  only  differences  being  in  degree  of  virulence,  mode 
of  onset,  and  portions  of  the  body  chiefly  affected. 

Sidney  Martin  has  recently  1  elaborated  the  views  an- 
nounced by  him  in  1892,  and  it  is  right  that  reference  should 
be  made  to  his  new  classification  of  bacterial  poisons.  This 
may  be  represented  as  follows : — 


1.  The  poisons  secreted  by  the  bacterium  itself 

= (ferment  ?  toxin  ?) 

2.  Products  of  digestive  action  of  bacterium  = 

albumoses  ; 

3.  Final  non-proteid  products=animal  alkaloid  ; 

4.  Poisons  present  in  the  bodies  of  the  bacillus 


=  Extracellular 
bacterial  poisons. 


=  Intracellular 
bacterial  poisons. 

The  poisons  of  bacteria  are,  according  to  Sidney  Martin, 
of  a  kind  which  cannot  be  fully  expressed  chemically,  but 
only  pathologically.  They  may  be  of  a  ferment  nature  in 
diphtheria  and  tetanus.  The  arguments  in  support  of  that 
view  are — (i)  that  they  act  in  infinitesimal  doses,  (2)  that 
they  may  act  slowly  and  produce  death  after  many  days  by 

1  Sidney  Martin,  M.D.,  F.R.S.,  F.R.C.P.,  Croonian  Lectures  delivered  before 
the  Royal  College  of  Physicians,  June,  1898. 


IMMUNITY  AND  ANTITOXINS  24$ 

profoundly  affecting  the  general  nutrition,  and  (3)  that  they 
are  sensitive  to  the  action  of  heat  in  a  way  that  no  chemical 
poisons  are  known  to  be.  If  they  are  considered  as  fer- 
ments, they  must  be  substances  which  have  a  peculiar  affin- 
ity for  certain  tissues  of  the  body  on  which  they  produce 
their  special  toxic  effect.  As  for  the  products  of  digestion, 
they  are  formed  either  by  the  bacillus  ingesting  the  proteid 
and  discharging  it  as  albumose,  or  the  digestion  occurs  by 
means  of  a  ferment  secreted  by  the  bacillus  in  the  body  of 
an  individual  or  animal  suffering  from  the  disease. 

Sidney  Martin  suggests  that  anthrax  produces  albumoses 
and  an  alkaloidal  substance,  the  former  producing  fever,  the 
latter  stupor.  In  tetanus  the  bacillus  produces  a  secretion 
of  the  bacillus  which  causes  the  convulsions.  The  albumoses 
present  in  this  disease  are  probably  due  to  the  secretory 
toxin.  In  diphtheria,  too,  we  have  a  secretory  poison  in 
the  membrane  and  in  the  tissues,  and  an  albumose  which  is 
possibly  the  result  of  the  secretion.  It  will  be  seen  that 
these  views  differ  in  some  particulars  from  those  to  which 
we  have  already  referred. 

However  the  details  of  the  modus  operandi  of  the  form- 
ation of  toxins  are  finally  settled,  we  know  that  there  comes 
a  time  when  the  disease  symptoms  vanish,  the  disease  de- 
clines, and  the  patient  recovers.  Many  of  the  older  schools 
of  medicine  explained  this  satisfactory  phenomenon  by 
saying  that  this  disease  exhausted  itself  after  having  "  gone 
through  "  the  body.  In  a  sense  that  idea  is  probably  true; 
but  recently  a  large  number  of  investigators  have  applied 
themselves  to  this  problem,  and  with  some  promising  results. 

Various  protective  inoculations  against  anthrax  were  prac- 
tised as  early  as  1881,  and  the  protected  animals  remained 
healthy.  In  1887  Wooldridge  succeeded  in  protecting  rab- 
bits from  anthrax  by  a  new  method,  by  which  he  showed 
that  the  growth  of  the  anthrax  bacillus  in  special  culture 
fluids  gave  rise  to  a  substance  which,  when  inoculated,  con- 


246  BACTERIA 

ferred  immunity.  In  1889  and  1890  Hankin  and  Ogata 
worked  at  the  subject,  and  announced  the  discovery  in  the 
blood  of  animals  which  had  died  of  anthrax  of  some  sub- 
stances which  appeared  to  have  an  antagonistic  and  neutral- 
ising effect  upon  the  toxins  of  anthrax  and  upon  the  anthrax 
bacilli  themselves.  These  substances,  they  afterwards 
found,  were  products  of  the  anthrax  bacillus.  Behring  and 
Kitasato  arrived  at  much  the  same  results  for  tetanus  and 
diphtheria.  The  next  step  was  to  isolate  these  substances, 
and,  separating  them  from  the  blood,  investigate  still  further 
their  constitution.  A  number  of  workers  were  soon  occu- 
pied at  this  task,  and  since  1891  Buchner,  Hankin,  the 
Klemperers,  Roux,  Sidney  Martin,  and  others  have  added 
to  our  knowledge  respecting  these  toxin-opposing  bodies 
known  as  antitoxins.  In  diphtheria,  as  we  have  seen,  the 
toxins  turned  out  to  be  soluble  bodies  allied  to  the  proteids, 
albumoses,  and  an  organic  acid.  Then  arose  the  question 
of  the  source  of  antitoxins.  Some  believed  they  were  a 
kind  of  ultratoxin — bodies  of  which  an  early  form  was  a 
toxin;  others  held  that,  as  the  toxins  were  products  of 
the  bacteria  invading  the  tissues,  the  antitoxins  were  of  the 
nature  of  ferments  produced  by  the  resisting  tissues. 
Finally,  they  came  to  be  looked  upon  as  protective  sub- 
stances produced  in  the  body  cells  as  a  result  of  toxin  action, 
and  held  in  solution  in  the  blood,  and  there  and  elsewhere 
exerting  their  influence  in  opposition  to  the  toxins.1  The 
progress  of  disease  is  therefore  a  struggle  between  the  toxins 

1  It  is  impossible  here  to  enter  into  a  detailed  consideration  of  the  various 
views  held  with  regard  to  the  formation  of  antitoxins.  It  is  needless  to  remark 
that  the  whole  matter  is  one  of  abstruse  technicality  and  intricacy.  These 
antitoxic  bodies  gradually  increase  in  the  blood  and  tissues,  and  their  action 
falls  into  two  groups  :  (a)  antitoxic,  which  counteract  the  effects  of  the  poison 
itself  ;  and  (ti)  antimierobic,  which  counteract  the  effects  of  the  bacillus  itself. 
"  In  one  and  the  same  animal  the  blood  may  contain  a  substance  or  substances 
which  are  both  antitoxic  and  antimierobic,  such,  for  example,  as  occurs  in  the 
process  of  the  formation  of  the  diphtheria  and  tetanus  antitoxic  serums  "  (Sid- 
ney Martin). 


IMMUNITY  AND  ANTITOXINS  24? 

and  the  antitoxins :  when  the  toxins  are  in  the  ascendency 
we  get  an  increase  of  the  disease ;  when  the  antitoxins  are 
in  the  ascendency  we  get  a  diminution  of  disease.  If  the 
toxins  triumph,  the  result  is  death;  if  the  antitoxins  and 
resistance  of  the  tissues  triumph,  the  result  is  recovery. 

We  may  now  consider  shortly  how  these  new  facts  were 
received  and  what  theories  of  explanation  were  put  forward 
to  explain  continued  insusceptibility  to  disease.  It  had  of 
course  been  known  for  a  long  time  past  that  one  attack  of 
small-pox,  for  example,  in  some  degree  protected  the  in- 
dividual from  a  subsequent  attack  of  the  same  disease.  To 
that  experience  it  was  now  necessary  to  add  a  large  mass 
of  experimental  evidence  with  regard  to  toxins  and  anti- 
toxins. The  theories  of  immunity  were  as  follows: 

1.  The  Exhaustion  Theory.     The  supporters  of  this  idea 
argued  that  bacteria  of  disease  circulating  in  the  body  ex- 
hausted the  body  of  the  supply  of  some  substance  or  condi- 
tion necessary  for  the  growth  and  development  of  their  own 
species. 

2.  The  Retention  Theory.     It  was  surmised  that  there  were 
certain  products  of  micro-organisms  of  disease  retained  in 
the  body  after  an  attack  which  acted  antagonistically  to  the 
further  growth  in  the  body  of  that  same  species. 

3.  The  Acquired  Tolerance  Theory.     Some  have  advanced 
the  theory  that,  after  a  certain  time,  the  human  tissues  ac- 
quired such  a  degree  of  tolerance  to  the  specific  bacteria  or 
their  specific  products  that  no  result  followed  their  action  in 
the  body.     The  tissues  become  acclimatised  to  the  disease. 

4.  The  Phagocyte  Theory.     This  theory,  which  gained  so 
many  adherents  when  first  promulgated  by  Metschnikoff, 
attributes  to  certain    cells   in    the   tissues   the   powers   of 
"  scavenging,"  overtaking  germs  of  disease,  and  absorbing 
them  into  their  own  protoplasm.     This,   indeed,   may  be 
actually  witnessed,  and  had  been  observed  before  the  time 
of  Metschnikoff.     But  it  was  he  who  applied  it  to  disease. 


248  BACTERIA 

He  came  to  the  conclusion  that  the  successful  resistance 
which  an  animal  offered  to  bacteria  depended  upon  the 
activity  of  these  scavenging  cells,  or  phagocytes.  These 
cells  are  derived  from  various  cellular  elements  normally 
present  in  the  body :  leucocytes,  endolethial  cells,  connect- 
ive-tissue corpuscles,  and  any  and  all  cells  in  the  body  which 
possess  the  power  of  ingesting  bacteria.  If  they  are  present 
in  large  numbers  and  active,  the  animal  is  insusceptible  to 
certain  diseases;  if  they  are  few  and  inactive,  the  animal  is 
susceptible. 

It  appears  that  the  bacteria  or  other  foreign  bodies  in  the 
blood  which  are  attacked  by  the  phagocyte  become  assimi- 
lated until  they  are  a  part  of  the  phagocyte  itself.  Met- 
schnikoff  explained  also  how  it  comes  to  pass  that  the 
phagocyte  is  able  to  encounter  bacteria  when  both  are  cir- 
culating through  the  blood.  It  is  guided  in  this  attack 
upon  the  organisms  by  a  power  termed  chcmiotaxis.  The 
bacteria  elaborate  a  chemical  substance  which  attracts  the 
phagocyte,  and  this  is  termed  "  positive  chemiotaxis. " 
But  it  may  occur  that  the  chemical  substance  produced  by 
the  bacteria  may  have  an  opposite,  or  repellent,  effect  upon 
the  leucocytes,  in  which  case  we  have  "  negative  chemio- 
taxis." a  It  is  not  to  be  wondered  at  that  such  a  theory  of 
immunity  based  upon  microscopical  observations,  should  at 
first  have  been  widely  accepted,  and  there  can  be  no  doubt 
that  Metschnikoff  has  collected  a  considerable  mass  of  evi- 
dence in  support  of  a  theory  of  phagocytosis.  But  when  it 
came  to  be  known  that  blood  serum,  from  which  all  leuco- 
cytes (phagocytes)  had  been  removed,  possessed  the  same 
immunising  effect  as  before,  it  was  clear  that  such  effect  was 
a  property  of  the  serum  per  se,  and  not  only  or  wholly  due 
to  the  scavenging  power  of  certain  cells  in  it.  Even  the 

1  Types  of  bodies  possessing  positive  chemiotaxis  for  bacteria  are  the  salts 
of  potassium,  peptone,  glycerine. 

2  Negative  chemiotaxis  is  illustrated  in  alcohol,  and  free  acids,  and  alkalies. 


IMMUNITY  AND   ANTITOXINS  249 

phagocyte  theory  depends  largely  for  its  validity  upon 
chemiotaxis,  which  latter  was  a  property  of  the  products  of 
the  bacteria  contained  in  the  blood  serum. 

5.  The  Antitoxin  Theory.  We  have  gathered,  then,  that 
whenever  bacteria,  introduced  into  the  blood  and  tissues, 
fail  to  multiply  or  produce  infection  (as  in  saprophytic 
bacteria,  or  in  immunity  of  a  particular  animal  from  a  specific 
microbe),  this  inability  to  perform  their  role  is  brought 
about  by  some  property  in  the  living  and  normal  blood 
serum  which  opposes  their  life  and  action ;  and  further  we 
have  learned  that  this  protective  property  is  exhaustible  ac- 
cording to  the  number  of  bacteria,  and  differs  with  various 
species  of  bacteria,  and  in  different  animals.  Buchner 
designates  these  protective  bodies,  held  in  solution  in  the 
blood,  alexines,  and  regards  them  as  belonging  to  the  albu- 
minous bodies  of  the  lymph  and  plasma.  Where  the  blood 
and  tissues  do  not  possess  this  power,  the  animal  is  suscept- 
ible. Now,  as  we  have  already  seen  from  the  experiments 
of  Ogata,  Kitasato,  and  others,  the  blood  of  an  animal  dead 
of  anthrax  is  protective  against  anthrax,  from  which  and 
the  foregoing  it  appears  that  microbes  produce  by  their 
growth  in  the  tissues  poisonous  substances  we  term  toxins, 
which  have  the  power  of  producing  in  the  blood  and  body 
cells  substances  inimical  to  themselves,  named  antitoxins, 
and  so  long  as  these  latter  substances  remain  in  the  tissues 
the  body  remains  insusceptible  to  further  attacks  of  the 
same  disease.  Alexines  are  naturally  produced  antitoxins; 
antitoxins  are  acquired  alexines.  Hence  we  have  the  well- 
known  terms  ' '  natural  ' '  and  ' '  acquired  immunity. ' '  Of  the 
former  we  have  already  spoken.  Acquired  immunity  is  a 
protection  not  belonging  to  the  tissues  of  individuals  natur- 
ally and  as  part  of  their  constitution,  but  it  is  acquired 
during  their  lives  as  a  further  accomplishment,  so  to  speak, 
of  their  tissues.  This  may  happen  in  one  or  both  of  two 
ways.  Either  it  may  be  an  involuntary  acquired  immunity, 


2$0  BACTERIA 

or    a    voluntary    acquired    immunity.     For   example,    the 
former  is  at  once  illustrated  by  an  attack  of  the  disease. 

Small-pox,  typhoid  fever,  even  scarlet  fever,  are  diseases 
which  very  rarely  attack  the  same  individual  twice.  That 
is  because  each  of  these  diseases  leaves  behind  it,  on  its  first 
appearance,  its  antitoxic  influence.  Hence  the  individual 
has  involuntarily  acquired  immunity  against  these  diseases. 
An  example  of  voluntary  acquired  immunity  is  also  at  hand 
in  the  old  method  of  preventive  inoculation  for  small-pox, 
or  variolation.  This  was  clearly  an  inoculation  setting  up 
an  artificial  and  mild  attack  of  small-pox,  by  which  the 
antitoxins  of  that  disease  were  produced,  and  protected  the 
individual  against  further  infection  of  small-pox ;  that  is  to 
say,  it  was  a  voluntary  acquired  immunity.  This  form  of 
artificial  production  of  protection  is  generally  called  art- 
ificial immunity.  Let  us  now  marshal  together  these  various 
terms  in  a  table  as  follows : 

j-  -,    •  \  =  a  condition  of  protection  of  insusceptibility  to  cer- 

1.  Natural  immunity  =  constitutional  protection  produced  by  alexines. 

2.  Acquired  immunity 

Acquired  naturally  (involuntarily)  produced  by  antitoxins  formed  by 

an  attack  of  the  disease. 
Acquired  artificially  (voluntary)= 

(a)  Active  immunity,  produced  by  direct  inoculation  of  the  weak- 

ened bacteria  or  weakened  toxins  of  the  disease,  e.  g.,  vac- 
cination, or  Pasteur's  treatment  of  rabies,  or  Haffkine's 
inoculation  for  cholera. 

(b)  Passive  immunity,  produced  by  inoculation,  not  of  the  disease 

or  of  its  toxins,  but  of  the  antitoxins  produced  in  the  body 
of  an  animal  suffering  from  the  specific  disease. 

It  is  hoped  that  previous  remarks  will  have  explained  the 
meaning  of  the  terms  used  in  the  above  table,  with  the 
exception  of  the  last  two  phrases  of  active  and  passive  im- 
munity. We  propose  now  to  consider  in  some  detail  the 
four  illustrations  quoted  under  these  two  headings,  viz., 


IMMUNITY  AND  ANTITOXINS  2$  I 

vaccination,  Pasteur's  treatment  of  rabies,  anti-cholera  in- 
oculation, and  antitoxin  inoculation.  From  all  accounts,  it 
is  to  be  feared  that  these  four  phases  of  artificial  immunity 
are  hopelessly  confused  in  the  educated  public  mind.  Nor 
is  this  to  be  wondered  at  when  we  reflect  upon  the  rapid 
growth  of  the  whole  science  of  immunity,  and  upon  the  ever- 
varying  forms  of  nomenclature  through  which  it  has  passed. 

Vaccination  for  Small-pox.  In  1717  Lady  Mary  Wortley 
Montagu  *  described  the  inoculation  of  small-pox  as  she  had 
seen  it  practised  in  Constantinople.  So  greatly  was  she  im- 
pressed with  the  efficacy  of  this  process  that  she  had  her 
own  son  inoculated  there,  and  in  1721  Mr.  Maitland,  a  sur- 
geon, inoculated  her  daughter  in  London.  This  was  the 
first  time  inoculation  was  openly  practised  in  England.2  For 
one  hundred  and  twenty  years  small-pox  inoculation  (or 
variolation,  as  it  is  more  correctly  termed)  was  practised  in 
England,  until  by  Act  of  Parliament  in  1840  it  was  pro- 
hibited. 

There  were  different  ways  of  performing  variolation,  but 
the  most  approved  method  was  similar  to  the  modern  system 
of  arm-to-arm  vaccination,  the  arm  being  inoculated  with  a 
lancet  in  one  or  more  places  with  small-pox  lymph  instead 
of,  as  now,  with  vaccine  lymph.  As  a  rule,  only  local  re- 
sults or  a  mild  attack  of  small-pox  followed,  which  prevented 
an  attack  of  natural  small-pox.  Its  disadvantage  is  apparent 
on  the  surface.  It  was  a  means  of  breeding  small-pox,  for 
the  inoculated  cases  were  liable  to  create  fresh  centres  of 
infection.  In  1796  Edward  Jenner,  who  was  a  country 
practitioner  in  Gloucestershire,  observed  that  those  persons 

1  The  friend  of  Addison  and   Pope,   who  married   Mr.    Edward   Wortley 
Montagu  in  1712,  and  on  his  appointment  to  the  ambassadorship  of  the  Porte 
in  1716  went  with  him  to  Constantinople.     They  remained  abroad  for  two 
years,  during  which  time  Lady  Wortley  Montagu  wrote  her  well-known  Let- 
ters to  her  sister  the  Countess  of  Mar,  Pope,  and  others. 

2  Crookshank,  History  and  Pathology  of  Vaccination. 


252  BACTERIA 

affected  with  cow-pox,  contracted  in  the  discharge  of  their 
duty  as  milkers,  did  not  contract  small-pox,  even  when 
placed  in  risk  of  infection.  Hence  he  inferred  that  inocul- 
ation of  this  mild  and  non-infectious  disease  would  be  pre- 
ferable to  the  process  of  variolation  then  so  widely  adopted 
in  England.  Jenner  therefore  suggested  the  substitution  of 
cow-pox  lymph  (vaccine)  in  place  of  small-pox  lymph,  as  in 
ordinary  variolation. 

It  should  not  be  forgotten  that  variolation  was  thus  the 
first  work  done  in  this  country  in  producing  artificial  im- 
munity, and  was  followed  by  vaccination,  which  was  only 
partly  understood.  Even  to-day  there  is  probably  much  to 
learn  respecting  it.  Both  variolation  and  vaccination  may 
be  described  as  active  immunisation  by  means  of  an  attenuated 
form  of  the  specific  virus  causing  the  disease.  The  nature  of 
the  specific  virus  of  both  small-pox  and  cow-pox  awaits  dis- 
covery. Burdon  Sanderson,  Crookshank,  Klein,  and  Cope- 
man  have  all  demonstrated  bacteria  in  cow-pox  or  vaccine 
lymph,  and  in  1898  Copeman  announced  that  he  had  isolated 
a  specific  bacillus  and  grown  it  upon  artificial  media.1 
Numerous  statements  have  been  made  to  the  effect  that  a 
specific  bacillus  has  been  found  in  small-pox  also.  But 
neither  in  small-pox  nor  cow-pox  is  the  nature  of  the  con- 
tagion really  known.2 

These  facts,  however,  do  not  remove  the  suspicion  which 
has  hitherto  rested  upon  vaccine  lymph  as  a  vehicle  for  bac- 
teria of  other  diseases  which  by  its  inoculation  may  thus  be 
contracted.  A  few  remarks  are  therefore  called  for  at  this 
juncture  upon  the  recent  work  of  Dr.  Monckton  Copeman 
and  Dr.  Frank  Blaxall  in  respect  to  what  is  known  zsglycer- 
inated  calf  lymph.  Evidence  has  been  forthcoming  to  sub- 

1  An  exhaustive  account  of  vaccine  may  be  found   in  the   Milroy  lectures 
delivered  in  1898  at  the  Royal  College  of  Physicians  by  S.  Monckton  Cope- 
man,  M.D. 

2  Crookshank,  Bacteriology  and  Infective  Diseases  ;  Virchow,   The  Huxley 
Lecture,  1898. 


IMMUNITY  AND  ANTITOXINS  2$$ 

stantiate  in  some  measure  the  distrust  which  many  of  the 
public  have  from  time  to  time  felt  in  the  vaccine  commonly 
used  in  vaccination,  hence  the  new  form  as  above  designated. 
This  retains  the  toxic  qualities  required  for  immunity,  but 
is  so  produced  that  it  possesses  in  addition  three  very 
important  advantages;  namely,  it  is  entirely  free  from 
extraneous  organisms,  it  is  available  for  a  large  number  of 
vaccinations,  and  it  retains  full  activity  for  eight  months. 
It  is  prepared  as  follows : 

A  calf,  aged  three  to  six  months,  is  kept  in  quarantine 
for  a  week.  If  then  found  upon  examination  to  be  quite 
healthy,  it  is  removed  to  the  vaccination  station,  and  the 
lower  part  of  its  abdomen  antiseptically  cleaned.  The 
animal  is  now  vaccinated  upon  this  sterilised  area  with 
glycerinated  calf  lymph.  After  five  days  the  part  is  again 
thoroughly  washed,  and  the  contents  of  the  vesicle,  which 
have  of  course  appeared  in  the  interval,  are  removed  with  a 
sterilised  sharp  spoon,  and  transferred  to  a  sterilised  bottle. 
This  is  now  removed  to  the  laboratory,  and  the  exact  weight 
of  the}  material  ascertained.  A  calf  thus  vaccinated  will 
yield  from  1 8  to  24  grams  of  vaccine  material.  This  is  now 
thoroughly  triturated  and  mixed  with  six  times  its  weight 
of  a  sterilised  solution  of  50  per  cent,  chemically  pure 
glycerine  in  distilled  water.  The  resulting  emulsion  is 
aseptically  stored  in  sealed  tubes  in  a  cool  place.  For  four 
weeks  it  is  carefully  examined  bacteriologically  until  the 
glycerine  has  absolutely  killed  any  possible  germ  that  may 
have  obtained  entrance.  When  by  agar  plates  it  is  demon- 
strably  sterile  it  is  ready  for  distribution. 

Pasteur 's  Treatment  of  Rabies.  Rabies  is  a  disease  affect- 
ing dogs  (in  Western  Europe)  and  wolves  (in  Russia),  and 
can  be  transmitted  to  other  animals  and  man,  infection  being 
carried  by  the  bite  of  a  rabid  animal.  It  takes  two  chief 
forms:  (i)  furious  rabies  and  (2)  paralytic  rabies.  The 
former  is  more  common  in  dogs.  The  animal  becomes  rest- 


254  BACTERIA 

less,  has  a  high-toned  bark,  and  snaps  at  various  objects. 
Sometimes  it  exhibits  depraved  appetite;  spasms  of  the 
throat  follow,  and  these  soon  develop  into  convulsions, 
which  are  followed  by  coma  and  death.  In  man  the  incub- 
ation period  is  fortunately  a  very  long  one,  averaging  about 
forty  days.  Nervous  irritability  is  the  first  sign;  spasms 
occur  in  the  respiratory  and  masticatory  muscles,  and  the 
termination  is  similar  to  rabies  in  the  dog.  The  symptom 
of  fear  of  water  is  a  herald  of  coming  fatality. 

Although  a  number  of  the  workers  at  the  Pasteur  Institute 
and  elsewhere  have  addressed  themselves  to  the  detection 
of  a  specific  microbe,  none  has  as  yet  been  found,  although, 
in  the  opinion  of  Pasteur,  such  an  agent  may  be  suspected 
as  the  cause. 

Pathologically  rabies  and  tetanus  (see  page  168)  are  closely 
allied  diseases,  and  the  recent  remarkable  additions  to  our 
knowledge  of  the  latter  disease  only  make  the  similarity 
more  evident.  There  are  in  rabies  three  chief  sets  of  post- 
mortem signs.  First,  and  by  far  the  most  important,  are  the 
changes  in  the  nervous  system.  Here  we  find  patches  of 
congestion  in  the  brain,  and  breaking  down  of  the  axis 
cylinders  of  the  nerves.  The  stomach,  in  the  second  place, 
exhibits  haemorrhagic  changes,  not  unlike  acute  arsencial 
poisoning.  Thirdly,  the  salivary  glands  show  a  degenerat- 
ive change  in  a  breaking  down  of  their  secreting  cells. 
Roux  has  pointed  out  that  in  life  the  saliva  of  a  mad  dog 
becomes  virulent  three  days  before  the  appearance  of  the 
symptoms  of  disease. 

We  may  now  turn  to  the  method  of  treatment  which  was 
introduced  by  Pasteur.  Before  his  time  cauterisation  of  the 
wound  was  the  only  means  adopted.  If  more  than  half  an 
hour  has  elapsed  since  the  bite,  cauterisation  is  of  little  or 
no  avail.  The  basis  for  Pasteur's  treatment  was  the  differ- 
ence in  virulence  obtainable  in  spinal  cords  infected  with 
rabies.  Pasteur  found  that  drying  the  cord  led  to  a  lessen- 


IMMUNITY  AND  ANTITOXINS 


255 


ing  of  its  virulence,  just  as  certain  other  conditions  increased 
its  virulence.  Next  he  established  the  fact  that  subcutane- 
ous injection  of  a  weak  virus,  followed  up  with  doses  of 
ever-increasingly  virulent  cords,  immunised  dogs  against 
infection  or  inoculation  of  fully  virulent  material.  From 
this  he  reasoned  that  if  he  could  establish  a  standard  of 
weakened  virulence  he  would  have  at  hand  the  necessary 
"  vaccine  "  for  the  treatment  of  the  disease. 

Subsequent  research  and  skilled  technique  resulted  in  a 
method  of  securing  this  standard,  which  he  found  to  be  a 
spinal  cord  dried  for  fourteen 
days.  The  exact  details  are  as 
follows:  The  spinal  cords  of  two 
rabbits  dead  of  rabies  are  removed 
from  the  spinal  canal  in  their  en- 
tirety by  means  of  snipping  the 
transverse  processes  of  the  ver- 
tebrae. Each  cord  is  divided  into 
three  more  or  less  equal  pieces, 
and  each  piece,  being  snared  by  a 
thread  of  sterilised  silk,  is  care- 
fully suspended  in  a  sterilised  glass 
jar.  At  the  bottom  of  the  jar 
is  a  layer,  about  half  an  inch 
deep,  of  sterilised  calcium 
chloride.  The  jars  are  then 
removed  to  a  dark  chamber, 
where  they  are  placed  at  a 
temperature  of  20-22°  C.  in 
wooden  cases.  Here  they 

are  left  to  dry.  Above  each  case  is  a  tube  of  broth,  to 
which  has  been  added  a  small  piece  of  the  corresponding 
cord,  in  order  to  test  for  any  organismal  element  that  may 
by  chance  be  included.  In  case  of  the  slightest  turbidity 
in  the  broth,  the  cord  is  rejected.  Fourteen  series  of  cords 


SUSPENDED  SPINAL  CORD 

In  drying  jar  containing  Calcium  Chloride 


256  BACTERIA 

are  thus  suspended  on  fourteen  consecutive  days.  The 
first,  second,  and  third  are  found  to  be  of  practically  equal 
virulence,  but  from  the  third  to  the  fourteenth  the  virulence 
proportionately  decreases,  and  on  the  fifteenth  day  the  cord 
would  be  practically  innocuous  and  non-virulent.  When 
treatment  is  to  be  commenced,  obviously  the  weakest — that 
is,  the  fourteenth  day — cord  is  used  to  make  the  "  vaccine," 
and  so  on  in  steadily  increasing  doses  (as  regards  virulence) 
up  to  and  including  a  third-day  cord.  The  fourteenth-day 
cord  is  therefore  taken,  and  a  small  piece  cut  off  and  macer- 
ated in  10  cc.  of  sterile  broth,  which  are  placed  in  a  conical 
glass  and  covered  with  two  layers  of  thick  filter-paper,  the 
glass  with  its  covering  having  been  previously  sterilised  by 
dry  heat.  When  the  patient  bitten  by  the  rabid  animal  is 
prepared,  3  cc.  of  this  broth  emulsion  of  spinal  cord  are  in- 
oculated by  means  of  a  hypodermic  needle  into  the  flanks 
or  abdominal  wall.  On  the  following  day  the  patient  returns 
for  an  inoculation  of  a  cord  of  the  thirteenth  day,  and  so  on 
until  a  rabid  cord  emulsion  of  the  first  three  days  has  been 
inoculated.  As  a  matter  of  practice,  the  dosage  depends 
upon  the  three  recognised  classes  of  bites,  viz.  (i)  bites 
through  clothing  (least  severe) ;  (2)  bites  on  the  bare  skin 
of  the  hand ;  (3)  bites  upon  the  face  or  head,  most  severe 
owing  to  the  vascularity  of  these  parts.  An  example  of 
each,  which  the  writer  was  permitted  to  take  in  the  Pasteur 
Institute,  may  be  here  added  to  make  quite  clear  the  entire 
practice.  (See  page  258.) 

It  may  be  well  to  add  the  returns  of  inoculation  made  at 
the  Pasteur  Institute,  Rue  Dutot,  Paris,  as  above  described. 
They  are  as  follows : 


Year. 
1886  

No.  of  Persons 
inoculated. 

.    2,671 

No.  of 
Deaths. 

25 

Rate  of 
Mortality. 

O.Q4 

l887.  . 

,    1.770 

O.7Q 

1888.. 

,    1.622 

0 

0.55 

IMMUNITY  AND  ANTITOXINS  257 


Year. 
1880 

No.  of  Persons 
inoculated. 

I  810 

No.  of 
Deaths. 

7 

Rate  of 
Mortality. 

o  78 

1890 

I  ^40 

/ 

O   "22 

1801.  . 

,     I   CCQ 

4 

u-3^ 

O  2? 

1802.  . 

,    i  700 

W"*D 
O  22 

1  80^ 

I  648 

6 

O  ?6 

1804.  . 

.  1,787 

7 

W'OV 

o.  so 

i8o^.  . 

.    I,  ^20 

2 

O.I"? 

1806.  . 

.    1,^08 

4 

O  7.O 

1807.. 

.      1.^21 

6 

0.70 

Pasteur's  treatment  of  rabies  by  inoculation  of  emulsions  of 
dried  spinal  cord  is,  therefore,  a  "  vaccination  "  of  attenu- 
ated virus,  resulting  in  antitoxin  formation,  to  the  further 
protection  of  the  individual  against  rabies. 

One  further  example  of  the  modern  application  of  the 
principles  of  active  acquired  immunity  may  be  shortly  men- 
tioned. We  refer  to  the  cholera  and  plague  vaccinations. 
The  vaccination  in  small-pox  is  an  inoculation  of  the  virus 
of  the  disease  ;  the  rabies  inoculation  is  a  transmission  of  the 
vital  products  of  the  disease  attenuated ;  the  plague  and 
cholera  vaccinations  are  inoculations  of  pure  cultures  of  living 
virus  from  outside  the  body.  Inoculating  cholera  virus 
against  cholera  has  been  made  illegal,  as  variolation  was 
in  1840.  But  Haffkine  has  prepared  two  vaccines.  The 
weak  one  is  made  from  pure  cultures  of  Koch's  spirillum  of 
Asiatic  cholera,  attenuated  by  growth  to  several  generations 
on  agar  or  broth  at  39°  C.  The  strong  one  is  from  similar 
culture  the  virulence  of  which  has  been  increased.  One 
cubic  centimetre  of  the  first  vaccine  is  injected  hypoder- 
mically  into  the  flank,  and  the  second  vaccine  three  or  four 
days  afterwards.  The  immunisation  is  prophylactic,  not 
remedial,  and  its  action  takes  effect  five  or  six  days  after 
the  second  vaccine  has  been  injected. 

In  plague  the  same  plan  has  been  followed.     Luxurious 


258 


BACTERIA 


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\D  -^  c<S  in  Tt  en 


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lOvO    t^OO     O  O     W 


IMMUNITY  AND  ANTITOXINS 

crops  of  Kitasato's  plague  bacillus  are  grown  on  ordinary 
nutritive  media  plus  large  quantities  of  fat.  The  fat  of 
milk,  as  clarified  butter,  is  that  generally  used.  Under  the 
globules  of  fat  flakes  of  culture  grow  like  stalactites,  hanging 
down  into  the  clear  broth.  These  are  in  time  shaken  to  the 
bottom,  and  a  second  crop  grows  on  the  under-surface  of  the 
fat.  In  the  course  of  a  month  perhaps  half  a  dozen  such 
crops  are  obtained  and  shaken  down  into  the  fluid,  until  the 
latter  assumes  an  opaque  milky  appearance.  This  is  now, 
unlike  the  cholera  vaccine,  exposed  to  a  temperature  of  70° 
C.,  by  which  the  microbes  are  killed.  The  culture  contains 
all  the  toxins,  and  the  dose  is  3  cc.  This  preparation  has 
the  advantage  of  being  easily  prepared,  obtainable  in  large 
quantities,  and  requires  no  animals  in  its  preparation. 
When  inoculated  it  produces  local  pain  and  swelling  at  the 
site  of  inoculation,  and  general  reactive  symptoms  such  as 
fever.  From  a  careful  analysis  of  the  results  of  this  inocu- 
lation, it  is  shown  that  the  efficacy  of  the  prophylactic  de- 
pends upon  the  virulence  of  the  bacillus  culture  from  which 
the  vaccine  is  prepared,  and  upon  its  dose  and  ability  to 
produce  a  well-marked  febrile  reaction.  It  appears  to  be 
more  effective  in  the  prevention  of  deaths  than  of  attacks. 

The  anti-typhoid  vaccination  is  another  example  of  in- 
oculation to  secure  active  immunity.  It  is  needless,  per- 
haps, to  point  out  that  all  these  vaccinations,  except  rabies, 
are  prophylactic,  and  not  curative. 

Passive  Immunity  ;  Preparation  of  Antitoxins.  We  must 
now  consider  the  question  of  passive  immunity.  This,  it 
will  be  remembered,  may  be  defined  as  a  protection  (against 
a  bacterial  disease)  produced  by  inoculation,  not  of  the  dis- 
ease itself,  as  in  small-pox  inoculation,  nor  yet  of  its  weak- 
ened toxins,  as  in  rabies,  but  of  the  antitoxins  produced  in 
the  body  of  an  animal  suffering  from  that  particular  disease. 
Examples  of  this  treatment  are  increasing  every  year,  and 
the  term  "  antitoxin  "  has  now  become  almost  a  household 


260  BACTERIA 

word.  The  chief  examples  are  to  be  found  in  diphtheria, 
tetanus,  streptococcus,  and  pneumococcus. 

To  be  of  value,  antitoxins  must  be  used  as  early  as  pos- 
sible, before  tissue  change  has  occurred  and  before  the 
toxins  have,  so  to  speak,  got  the  upper  hand.  When  the 
toxins  are  in  the  ascendency  the  patient  suffers  more  and 
more  acutely,  and  may  succumb  before  there  has  been  time 
for  the  formation  in  his  own  body  of  the  antitoxins.  If  he 
can  be  tided  over  the  "  crisis,"  theoretically  all  will  be  well, 
because  then  his  own  antitoxin  will  eventually  gain  the 
upper  hand.  But  in  the  meantime,  before  that  condition 
of  affairs,  the  only  way  is  to  inject  antitoxins  prepared  in 
some  animal's  tissues  whose  disease  began  at  an  earlier  date, 
and  thus  add  antitoxins  to  the  blood  of  our  patient,  early 
in  the  disease,  and  the  earlier  the  better,  for,  however  soon 
this  is  done,  it  is  obvious  that  the  toxins  begin  their  work 
earlier  still.  It  should  not  be  necessary  to  add  that  general 
treatment  must  also  be  continued,  and  indeed  local  germ- 
icidal  treatment,  e.g.,  of  the  throat  in  diphtheria  and  the 
poisoned  wound  in  tetanus.  Further,  in  a  mixed  infection, 
as  in  glandular  abscesses  with  diphtheria,  it  must  be  borne 
in  mind  that  the  antitoxin  is  specific  and  may  therefore 
probably  fail  in  such  mixed  cases. 

After  these  preliminary  remarks  we  will  now  consider 
shortly  some  of  the  methods  employed  for  the  production 
of  antitoxins.  An  animal  is  required  from  whose  body  a 
considerable  quantity  of  blood  can  be  drawn  without  injur- 
ious effect.  Moreover,  it  must  be  an  animal  that  can  stand 
an  attack  of  such  diseases  as  diphtheria  and  tetanus.  Such 
an  animal  is  the  horse.  Now,  by  injecting  into  the  horse 
(a)  living  organisms  of  the  specific  disease,  but  in  non-fatal 
doses,  or  (b)  dead  cultures,  or  (c)  filtered  cultures  containing 
no  bacteria  and  only  the  toxins,  we  are  able  to  produce  in 
the  blood  of  the  horse  first  the  toxins  and  then  the  antitoxins 
of  the  disease  in  question.  The  non-poisonous  doses  of 


IMMUNITY  AND  ANTITOXINS 


26l 


r 


living  organisms  can  be  weakened,  or,  as  we  say,  attenuated, 
by  various  means.  Dead  cultures  have  not  been  much  used 
to  produce  immunity  except  by  Pfeiffer.  In  actual  practice 
the  third  method  is  much  the  most  general,  viz.,  filtering  a 
fluid  culture  free  from  the  bacteria,  and  then  inoculating 
this  in  ever-increasing  doses.  The  preparation  of  diphtheria 
antitoxin  may  be  taken  as  an  example,  but  what  follows 
would  be  equally  applicable  to  other  diseases,  such  as  tetanus. 

1.  To  Obtain  the  Toxin.     First  grow  a  pure  culture  of  the 
Klebs-Loffler  bacillus  of  diphtheria  in  large  flasks  contain- 
ing "  Loffler's  medium,"   or  a  solution   made  by  mixing 
three  parts  of  blood  serum  with  one  of  beef  broth  and  add- 
ing one  per  cent,  of  common  salt  (Na  Cl)  and  one  per  cent, 
of  peptone.     An  alkaline  medium  is  preferable.     The  flask 
was  thoroughly  sterilised  before  use,  and  is 

now  plugged  with  sterile  cotton-wool  and  in- 
cubated at  77°  C.  for  three  or  four  weeks. 
Pure  air  may  be  passed   over   the   culture 
periodically,     thereby    aiding    the    growth. 
After  the  lapse  of  about  a  month  a  scum  of 
diphtheria  growth  will  have  appeared 
over  the  surface  of  the  fluid.     This  is 
now  filtered  into  sterilised  flasks,  and 
some  favourable  antiseptic  added  to 
ensure   that   nothing    foreign    to    the 

i      ,  1    a  .    t  j    j_i         ni  Flask  used  for  the  Preparation  of 

toxin  shall  flourish,  and  the  flasks  are       the  Toxin  of  Diphtheria 

kept    in    the   dark.      Here,    then,   we 

have  the  product,  the  toxin,  ready  for  injection  into  the  horse. 

2.  Immunisation  of  the  Horse.     It  is  evident  that  only 
healthy  horses  are  of  service  in  providing  healthy  antitoxin, 
even  as  healthy  children  are  necessary  in  arm-to-arm  vacci- 
nation.    To  provide  against  any  serious  taint  the  horse  is 
tested  for  glanders  (with  mallein)  and  for  tuberculosis  (with 
tuberculin).     The  dose  of  the  injection  of  toxin  is  at  the 
commencement  about       cc.,  or  a  little  more.     The  site  of 


262  BACTERIA 

the  inoculation  is  the  apex  of  the  shoulder,  which  has  been 
antiseptically  cleaned.  A  mere  prick  is  the  whole  operation. 
After  the  first  injection  there  is  generally  a  definite  febrile 
reaction  and  a  slight  local  swelling.  From  ^  or  £  cc.  the 
dose  is  steadily  increased,  until  at  the  end  of  two  or  three 
months '  perhaps  as  much  as  300  cc.  (or  even  half  a  litre) 
may  be  injected  without  causing  the  reaction  which  the 
initial  injection  of  -J^  cc.  caused  at  the  outset.  This  shows 
an  acquired  tolerance  of  the  tissues  of  the  horse  to  the  toxic 
material.  After  injecting  500  cc.  into  the  horse  without 
bad  effect,  the  animal  has  a  rest  of  four  or  five  days. 

3.  To  Obtain  the  Antitoxin.  During  this  period  of  rest 
the  interaction  between  the  living  body  cells  of  the  animal 
and  the  toxins  results  in  the  production  in  the  blood  of  an 
antitoxin.  By  means  of  a  small  sterilised  cannula,  five,  or 
eight,  or  even  ten  litres  of  blood  are  drawn  from  the  jugu- 
lar vein  of  the  horse  into  sterilised  flasks  or  jars.  The  top 
of  the  jar  is  closed  by  two  paper  coverings  before  it  is 
sterilised.  Then  it  is  again  covered  with  a  further  loose 
one.  Before  use  the  loose  one  is  removed  and  replaced  by 
a  metal  (zinc)  lid,  which  has  been  separately  sterilised.  This 
metal  lid  contains  an  aperture  large  enough  for  the  tube 
which  conveys  the  blood  from  the  cannula  to  pass  through. 
The  tube,  therefore,  passes  through  the  metal  lid  and  two 
paper  covers,  which  it  was  made  to  pierce.  When  enough 
blood  has  passed  into  the  vessel  the  tube  is  withdrawn,  and 
the  metal  lid  slightly  turned.  Thus  the  contained  blood  is 
protected  from  the  air.2 

1  To  shorten  this  period  Dr.  Cartwright  Wood  has  adopted  a  plan  by  which 
time  may  be  saved,  and  200  cc.  injected  say  within  the  first  two  or  three  weeks. 
This  is  accomplished  by  using  a  "  serum  toxin  "  (containing  albumoses,  but  not 
ferments)  previously  to  the  broth  toxin,  an  ingenious  method  which  we  cannot 
enter  into  here. 

2  At  the  conclusion  of  the  operation  the  cannula  is  removed  from  the  jugular 
vein,  and  the  wound  is  closed  by  the  valvular  character  of  the  slit  in  the  skin 
and  vein  and  the  elasticity  of  the  wall  of  the  vein.     No  stitching  or  dressing 
is  required.     Indeed,  it  is  striking  to  observe  in  the  horse  an  entire  absence  of 
pain  throughout  the  proceedings. 


IMMUNITY  AND  ANTITOXINS  263 

The  jar  containing  the  blood  (which  contains  the  anti- 
toxin) is  next  placed  in  a  dark,  cool  cellar,  where  it  stands 
for  two  or  three  days.  During  this  time  the  blood  naturally 
coagulates,  the  corpuscles  falling  as  a  dense  clot  to  the  bot- 
tom, and  the  faintly  yellow  serum  rising  to  the  top.  The 
serum,  or  liquor  sanguinis,  averages  about  50  per  cent,  of 
the  total  blood  taken.  Sometimes  antiseptics  are  added 
with  a  view  to  preservation.  It  is  generally  filtered  before 
bottling  for  therapeutic  use,  and  sometimes  examined 
bacteriologically  as  a  test  of  purity. 

4.  The  Use  of  Antitoxins.  The  antitoxins  are  now  ready 
for  injection  into  the  patient  who  has  contracted  diphtheria, 
and  in  whose  blood  toxins  are  in  the  ascendency  and  under 
which  the  individual  may  succumb.  They  are  injected  in 
varying  doses,  as  we  have  already  pointed  out.1  The  gen- 
eral result  is  that  mortality  has  been  greatly  lessened, 
and  that  in  fatal  cases  there  has  been  a  considerable  length- 
ening of  the  period  of  life.  Moreover,  the  whole  clinical 
course  of  the  disease  has  been  greatly  modified,  and  suffer- 
ing lessened.2 

1  The  term  unit  is  used  as  a  standard  measurement.     This  means  the  amount 
of  antitoxin  which  will  just  neutralise  ten  times  the  minimum  fatal  dose  of  the 
toxin  in  a  guinea-pig  (250  grams  toxin  to  kill  on  the  fourth  day).     If  I  cc.  of 
the  antitoxic  serum  is  required  for  this,  one  unit  is  contained  in  I  cc.;  if  o.oi 
cc.  is  sufficient,  then  100  units  are  contained  in  the  cc.     Not  less  than  1500 
units  should  be  administered  for  a  dose,   and  repeated  every  twelve  hours. 
In  severe  cases  two  or  three  times  this  amount  may  be  given. 

2  The  value  of  antitoxin  treatment  in  diphtheria  is  discussed  in  the  Brit. 
Med.  Jour.,  1899,  pp.  197  and  268,  by  E.  W.  Goodall,  M.D. 


CHAPTER  VIII 
BACTERIA  AND  DISEASE 

PROBABLY  the  most  universally  known  fact  respecting 
bacteria  is  that  they  are  related  in  some  way  to  the 
production  of  disease.  Yet  we  have  seen  that  it  was  not  as 
disease-producing  agents  that  they  were  first  studied.  In- 
deed, it  is  only  within  comparatively  the  latest  period  of  the 
two  centuries  during  which  they  have  been  more  or  less 
under  observation  that  our  knowledge  of  them  as  causes  of 
disease  has  assumed  any  exactitude  or  general  recognition. 
Nor  is  this  surprising,  for  although  an  intimate  relationship 
between  fermentation  and  disease  had  been  hinted  at  in  the 
middle  of  the  seventeenth  century,  it  was  not  till  the  time 
of  Pasteur  that  the  bacterial  cause  of  fermentation  was  ex- 
perimentally and  finally  established. 

In  the  middle  of  the  seventeenth  century  men  learned, 
through  the  eyes  of  Leeuwenhoek,  that  drops  of  water  con- 
tained "  moving  animalcules."  A  hundred  years  later 
Spallanzani  demonstrated  the  fact  that  putrefaction  and 
fermentation  were  set  up  in  boiled  vegetable  infusions  when 
outside  air  was  admitted,  but  when  it  was  withheld  from 
these  boiled  infusions  no  such  change  occurred.  Almost  a 
hundred  years  more  passed  before  the  epoch-making  work 
of  Tyndall  and  Pasteur,  who  separated  these  putrefactive 
germs  from  the  air.  Quickly  following  in  their  footsteps 
came  Davaine  and  Pollender,  who  found  in  the  blood  of 
animals  suffering  from  anthrax  the  now  well-known  specific 

264 


BACTERIA   AND  DISEASE  26$ 

and  causal  bacillus  of  that  disease.  Improvements  in  the 
microscope  and  in  methods  of  cultivation  (Koch's  plate 
method  in  particular)  soon  brought  an  army  of  zealous  in- 
vestigators into  the  field,  and  during  the  last  twenty  years 
first  this  disease  and  then  that  have  been  traced  to  a  bac- 
terial origin.  We  may  summarise  the  vast  mass  of  histori- 
cal, physiological,  and  pathological  research  extending  from 
1650  to  1898  in  three  great  periods:  the  period  of  detection 
of  living,  moving  cells  (Leeuwenhoek  and  others  in  the 
seventeenth  century) ;  the  period  of  the  discovery  of  their 
close  relationship  to  fermentation  and  putrefaction  (Spallan- 
zani,  Schulze,  Schwann,  in  the  eighteenth  century);  and, 
thirdly,  the  period  of  appreciation  of  the  role  of  bacteria  in 
the  economy  of  nature  and  in  the  production  of  disease 
(Tyndall,  Pasteur,  Lister,  Koch,  in  the  nineteenth). 

But  we  must  look  less  cursorily  at  the  growth  of  the  idea 
of  bacteria  causing  disease.  More  than  two  hundred  years 
ago  Robert  Boyle  (1627-91),  the  philosopher,  who  did  so 
much  towards  the  foundation  of  the  present  Royal  Society, 
wrote  a  learned  treatise  on  The  Pathological  Part  of  Physic. 
He  was  one  of  the  earliest  scientists  to  declare  that  a  re- 
lationship existed  between  fermentation  and  disease.  When 
more  accurate  knowledge  was  attained  respecting  ferment- 
ation, great  advance  was  consequently  made  in  the  etiology 
of  disease.  The  preliminary  discoveries  of  Fuchs  and  others 
between  1840  and  1850  had  relation  to  the  existence  in  dis- 
eased tissues  of  a  large  number  of  bacteria.  But  this  was 
no  proof  that  such  germs  caused  such  diseases.  It  was  not 
till  Davaine  had  inoculated  healthy  animals  with  bacilli  from 
the  blood  of  an  anthrax  carcass,  and  had  thus  produced  the 
disease,  that  reliance  could  be  placed  upon  that  bacillus  as 
the  vera  causa  of  anthrax.  Too  much  emphasis  cannot  be 
laid  upon  this  idea,  that  unless  a  certain  organism  produces 
in  healthy  tissues  the  disease  in  question,  it  cannot  be  con- 
sidered as  proven  that  the  particular  organism  is  related  to 


266  BACTERIA 

the  disease  as  cause  to  effect.  In  order  to  secure  a  standard 
by  which  all  investigators  should  test  their  results,  Koch  in- 
troduced four  postulates.  Until  each  of  the  four  has  been 
fulfilled,  the  final  conclusion  respecting  the  causal  agent 
must  be  considered  sub  judice.  The  postulates  are  as 
follows : 

(a)  The  organism  must  be  demonstrated  in  the  circulation 
or  tissues  of  the  diseased  animal. 

(b)  The  organism  thus  demonstrated  must  be  cultivated 
in  artificial  media  outside  the  body,  and  successive  genera- 
tions of  &pure  culture  of  that  organism  must  be  obtained. 

(c)  Such   pure    cultures    must,    when    introduced   into  a 
healthy  and  susceptible  animal,  produce  the  specific  disease. 

(d)  The  organism  must  be  found  and  isolated  from  the 
circulation  or  tissues  of  the  inoculated  animal. 

It  is  evident  that  there  are  some  diseases — for  example, 
cholera,  leprosy,  and  typhoid — which  are  not  communicable 
to  lower  animals,  and  therefore  their  virus  cannot  be  made 
to  fulfil  postulate  (c).  In  such  cases  there  is  no  choice. 
They  cannot  be  classified  along  with  tubercle  and  anthrax. 
Bacteriologists  have  little  doubt  that  Hansen's  bacillus  of 
leprosy  is  the  cause  of  that  disease,  yet  it  has  not  fulfilled 
postulates  (&)  and  (c).  Nor  has  the  generally  accepted 
bacillus  of  typhoid  fulfilled  postulate  (c),  yet  by  the  major- 
ity it  is  provisionally  accepted  as  the  agent  in  producing 
typhoid.  Hence  it  will  be  seen  that,  though  there  is  an 
academical  classification  of  causal  pathogenic  bacteria  ac- 
cording as  they  respond  to  Koch's  postulates,  yet  neverthe- 
less, there  are  a  number  of  pathogenic  bacteria  which  are 
looked  upon  as  causes  of  disease  provisionally.  Anthrax 
and  tubercle,  with  perhaps  the  organisms  of  suppuration, 
tetanus,  plague,  and  actinomycosis,  stand  in  the  first  order 
of  pathogenic  germs.  Then  comes  a  group  awaiting 
further  confirmation.  It  includes  the  organisms  related  to 
typhoid,  cholera,  malaria,  leprosy,  diarrhoea, and  pneumonia. 


BACTERIA   AND  DISEASE  267 

Then  comes  in  a  third  category,  a  long  list  of  diseases,  such 
as  scarlet  fever,  small-pox,  rabies,  and  others  too  numerous 
to  mention,  in  which  the  nature  of  the  causal  agent  is  still 
unknown.  Hence  it  must  not  be  supposed  that  every  dis- 
ease has  its  germ,  and  without  a  germ  there  is  no  disease. 
Such  universal  assertions,  though  not  uncommonly  heard, 
are  devoid  of  accuracy. 

In  the  production  of  bacterial  disease  there  are  two  factors. 
First,  there  is  the  body  tissue  of  the  individual;  secondly, 
there  is  the  specific  organism. 

Whatever  may  be  said  hereinafter  with  regard  to  the 
power  of  micro-organisms  to  cause  disease,  we  must  under- 
stand one  cardinal  point,  namely,  that  bacteria  are  never 
more  than  causes,  for  the  nature  of  disease  depends  upon  the 
behaviour  of  the  organs  or  tissues  ^vith  which  the  bacteria  or 
their  products  meet  (Virchow).  Fortunately  for  a  clear  con- 
ception of  what  "  organs  and  tissues  "  mean,  these  have 
been  reduced  to  a  common  denominator,  the  cell.  Every 
living  organism,  of  whatever  size  or  kind,  and  every  organ 
and  tissue  in  that  living  organism,  contains  and  consists  of 
cells.  Further,  these  cells  are  composed  of  organic  chemical 
substances  which  are  not  themselves  alive,  but  the  mechani- 
cal arrangement  of  which  determines  the  direction  and  power 
of  their  organic  activity  and  of  their  resistance  to  the  specific 
agents  of  disease.  With  these  facts  clearly  before  us,  we 
may  hope  to  gain  some  insight  into  the  reasons  for  depart- 
ure from  health. 

The  normal  living  tissues  have  an  inimical  effect  upon 
bacteria.  Saprophytic  bacteria  of  various  kinds  are  normally 
present  on  exposed  surfaces  of  skin  or  mucous  membrane. 
Tissues  also  which  are  dead  or  depressed  in  vitality  from 
injury  or  previous  disease,  but  which  are  still  in  contact 
with  the  tissues,  afford  an  excellent  nidus  for  the  growth  of 
bacteria.  Still  these  have  not  the  power,  unless  specific,  to 
thrive  in  the  normal  living  tissue.  It  has  been  definitely 


268  BACTERIA 

shown  that  the  blood  fluids  of  the  body  have  in  their  fresh 
state  the  germicidal  power  (alexines)  which  prevents  bacteria 
from  flourishing  in  them.  Such  action  does  undoubtedly 
depend  in  measure  upon  the  number  of  germs  as  well  as 
their  quality,  for  the  killing  power  of  blood  and  lymph  must 
be  limited.  Buchner  has  pointed  out  that  the  antagonistic 
action  of  these  fluids  depends  in  part  possibly  upon  pha- 
gocytosis, but  largely  upon  a  chemical  condition  of  the 
serum.  The  blood,  then,  is  no  friend  to  intruding  bacteria. 
Its  efforts  are  to  a  certain  extent  seconded  by  the  lymphoid 
tissue  throughout  the  body.  Rings  of  lymphoid  tissue 
surround  the  oral  openings  of  the  trachea  (windpipe)  and 
oesophagus  (gullet);  the  tonsils  are  masses  of  lymphoid 
tissue.  Composed  as  it  is  of  cells  having  a  germicidal  in- 
fluence when  in  health,  the  lymphoid  tissue  may  afford 
formidable  obstruction  to  intruding  germs. 

All  the  foregoing  points  in  one  direction,  namely,  that  if 
the  tissues  are  maintained  in  sound  health,  they  form  a  very 
resistant  barrier  against  bacteria.  But  we  know  from  ex- 
perience that  a  full  measure  of  health  is  not  often  the  happy 
condition  of  human  tissues ;  we  have,  in  short,  a  variety  of 
circumstances  which,  as  we  say,  predispose  the  individual  to 
disease.  One  of  the  commonest  forms  of  predisposition  is 
that  due  to  heredity.  Probably  it  is  true  that  what  are 
known  as  hereditary  diseases  are  due  far  more  to  a  hereditary 
predisposition  than  to  any  transmission  of  the  virus  itself  in 
any  form.  Antecedent  disease  predisposes  the  tissues  to  form 
a  nidus  for  bacteria ;  conditions  of  environment  or  personal 
habits  frequently  act  in  the  same  way.  Damp  soils  must  be 
held  responsible  for  many  disasters  to  health,  not  directly, 
but  indirectly,  by  predisposition  ;  dusty  trades  and  injurious 
occupations  have  a  similar  effect.  Any  one  of  these  three 
different  influences  may  in  a  variety  of  ways  affect  the 
tissues  and  increase  their  susceptibility  to  disease.  Not  in- 
frequently we  may  get  them  combined.  For  example,  the 


BACTERIA    AND   DISEASE  269 

following  is  not  an  unlikely  series  of  events  terminating  in 
consumption  (tuberculosis  of  the  lungs) : — (a)  The  individual 
is  predisposed  by  inheritance  to  tuberculosis ;  (b)  an  ordinary 
chronic  catarrh,  which  lowers  the  resisting  power  of  the 
lungs,  may  be  contracted;  (c)  the  epithelial  collections  in 
the  air  vesicles  of  the  lung — i.  e.,  dead  matter  attached  to 
the  body — afford  an  excellent  nidus  for  bacteria;  (d)  owing 
to  occupation,  or  personal  habits,  or  surroundings,  the 
patient  comes  within  a  range  of  tubercular  infection,  and 
the  specific  bacilli  of  tubercle  gain  access  to  the  lungs.  The 
result,  it  is  needless  to  state,  will  be  a  case  of  consumption 
more  or  less  acute  according  to  environment  and  treatment. 
The  channels  of  infection  by  which  organisms  gain  the 
vantage-ground  afforded  by  the  depressed  tissues  are  various, 
and  next  to  the  maintenance  of  resistant  tissues  they  call  for 
most  attention  from  the  physician  and  surgeon.  It  is  in 
this  field  of  preventive  medicine — that  is  to  say,  preventing 
infective  matter  from  ever  entering  the  tissues  at  all — that 
science  has  triumphed  in  recent  years.  It  is,  in  short,  ap- 
plied bacteriology,  and  therefore  claims  consideration  in  this 
place. 

1.  Pure  Heredity.     By  this  term  may  be  understood  the 
actual  transmission  from  the  mother  to  the  unborn  child  of 
the  specific  virus  of  the  disease.     That  such  a  conveyance 
may  occur  is  generally  admitted  by  pathologists,  but  it  is 
impossible  to  enter  fully  into  the  matter  in  such  a  book  as 
the  present.     Summarily  we  may  say  that,  though  this  sort 
of  transmission  is  possible,  it  is  not  frequent,  nor  is  disease 
appreciably  spread  through  such  a  channel.     Sixty  per  cent, 
of  consumptives,  it  has  been  estimated,  have  tuberculous 
progenitors,  and  this  is  the  highest  figure.     Many  would  be 
justified  from  experience  in  placing  it  at  half  that  number. 

2.  Inoculation,  or  inserting  virus  through  a  broken  surface 
of  skin,  is  itself  a  sufficiently  obvious  mode  of  infection  to 
call  for  little  comment.     Yet  it  is  under  this  heading  that 


2/0  BACTERIA 

a  word  must  be  said  of  that  remarkable  application  of  pre- 
ventive medicine  known  as  the  antiseptic  treatment  of 
wounds.  When  Lord  Lister  was  Professor  of  Surgery  in 
Glasgow,  he  was  impressed  with  the  greatness  of  the  evil  of 
putrefaction  in  wounds,  which  was  caused,  not  by  the 
oxygen  of  the  air,  as  Liebig  had  declared,  but  by  the  en- 
trance into  the  wound  of  fermentative  organisms  from  the 
air.  This  was  demonstrated  by  Pasteur,  who  pointed  out 
that  they  could  not  arise  de  novo  in  the  wound.  Hence  it 
appeared  to  Lister  that  these  fermentative  bacteria  which 
produce  putrefaction  in  wounds  must  either  be  kept  out  of 
the  wound  altogether,  or  killed,  or  their  action  prevented, 
in  the  wound.  To  keep  air  away  from  wounds  is  an  almost 
impossible  task,  and  thus  it  came  about  that  wounds  were 
dressed  with  a  solution  of  carbolic  acid. 

From  time  to  time  examples  occur  of  bacterial  disease 
being  directly  inoculated  in  wounds  made  with  polluted  in- 
struments, or  in  cuts  made  by  contaminated  broken  glass, 
or  in  gunshot  wounds.  Tetanus  is,  of  course,  one  of  the 
most  marked  examples. 

3.  Contagion  is  a  term  which  has  suffered  from  the  many 
ways  in  which  it  has  been  used.     Defined  shortly  and  most 
simply,  we  should  say  a  disease  is  contagious  when  it  can  be 
"  caught"  by  contact,  through  the  unbroken  surfaces,  be- 
tween   diseased    and    healthy    persons.      Ringworm    is   an 
example,  and  there  are  many  others. 

4.  The  Alimentary   Canal:    Food.      The   recent   Royal 
Commission  on  Tuberculosis  has  collected  a  large  mass  of 
evidence  in  support  of  the  view  that  tubercle  may  be  spread 
by  articles  of  food.     Milk  and  meat  from  tuberculous  animals 
naturally  come  in  for  the  largest  amount  of  condemnation. 
To  these  matters  we  refer  elsewhere. 

5.  The  Respiratory  Tract :  Air.     The  air  may  become  in- 
fected   with    germs    of   disease    from    dusty   trades,    dried 
sputum,  etc.     If  such  infected  air  be  inhaled,  pathogenic 


BACTERIA   AND  DISEASE  2JI 

results  will  follow,  especially  if  the  bacteria  are  present  in 
sufficient  numbers,  or  meet  with  devitalised,  and  therefore 
non-resisting,  tissues. 

These,  then,  are  the  five  possible  ways  in  which  germs 
gain  access  to  the  body  tissues.  The  question  now  arises, 
How  do  bacteria,  having  obtained  entrance,  set  up  the  pro- 
cess of  disease  ?  For  a  long  time  pathologists  looked  upon 
the  action  of  these  microscopic  parasites  in  the  body  as 
similar  to,  if  not  identical  with,  the  larger  parasites  some- 
times infesting  the  human  body.  Their  work  was  viewed 
as  a  devouring  of  the  tissues  of  the  body.  Now,  it  is  well 
known  that,  however  much  or  little  of  this  may  be  done, 
the  specific  action  of  pathogenic  bacteria  is  of  a  different 
nature.  It  is  twofold.  We  have  the  action  of  the  bacteria 
themselves,  and  also  of  their  products  or  toxins.  In  par- 
ticular diseases,  now  one  and  now  the  other  property  comes 
to  the  front.  In  bacterial  diseases  affecting  or  being  trans- 
mitted mostly  by  the  blood,  it  is  the  toxins  which  act 
chiefly.  The  convenient  term  infection  is  applied  to  those 
conditions  in  which  there  has  been  a  multiplication  of  living 
organisms  after  they  have  entered  the  body,  the  word  in- 
toxication indicating  a  condition  of  poisoning  brought  about 
by  their  products.  It  will  be  apparent  at  once  that  we  may 
have  both  these  conditions  present,  the  former  before  the 
latter,  and  the  latter  following  as  a  direct  effect  of  the 
former.  Until  intoxication  occurs  there  may  be  few  or  no 
symptoms,  but  directly  enough  bacteria  are  present  to  pro- 
duce in  the  body  certain  poisons  in  sufficient  amount  to 
result  in  more  or  less  marked  tissue  change,  then  the  symp- 
toms of  that  tissue  change  appear.  This  period  of  latency 
between  infection  and  the  appearance  of  the  disease  is 
known  as  the  incubation  period.  Take  typhoid,  for  example. 
A  man  drinks  a  typhoid-polluted  water.  For  about  four- 
teen days  the  bacilli  are  making  headway  in  his  body  with- 
out his  being  aware  of  it.  But  at  the  end  of  that  incubation 


2/2  BACTERIA 

period  the  signs  of  the  disease  assert  themselves.  Professor 
Watson  Cheyne  and  others  have  maintained  that  there  is 
some  exact  proportion  between  the  number  of  bacteria 
gaining  entrance  and  the  length  of  the  incubation  period. 

Speaking  generally,  we  may  note  that  pathogenic  bacteria 
divide  themselves  into  two  groups:  those  which,  on  enter- 
ing the  body,  pass  at  once,  by  the  lymph  or  blood  stream, 
to  all  parts  of  the  body,  and  become  more  and  more  diffused 
throughout  the  blood  and  tissues,  although  in  some  cases 
they  settle  down  in  some  spot  remote  from  the  point  of  en- 
trance, and  produce  their  chief  lesions  there.  Tubercle  and 
anthrax  would  be  types  of  this  group.  On  the  other  hand, 
there  is  a  second  group,  which  remain  almost  absolutely 
local,  producing  only  little  reaction  around  them,  never 
passing  through  the  body  generally,  and  yet  influencing  the 
whole  body  eventually  by  means  of  their  ferments  or  toxins. 
Of  such  the  best  representatives  are  tetanus  and  diphtheria. 
The  local  site  of  the  bacteria  is,  in  this  case,  the  local  man- 
ufactory of  the  disease. 

Whilst  the  mere  bodily  presence  of  bacteria  may  have 
mechanical  influence  injurious  to  the  tissues  (as  in  the  small 
peripheral  capillaries  in  anthrax),  or  may  in  some  way  act 
as  a  foreign  body  and  be  a  focus  of  inflammation  (as  in 
tubercle),  the  real  disease-producing  action  of  pathogenic 
bacteria  depends  upon  the  chemical  poisons  (toxins)  formed 
directly  or  indirectly  by  them.  Though  within  recent  years 
a  great  deal  of  knowledge  has  been  acquired  about  the 
formation  of  these  bodies,  their  exact  nature  is  not  known. 
They  are  allied  to  albuminous  bodies  and  proteoses,  and  are 
frequently  described  as  tox-albumens.  It  may  be  found, 
after  all,  that  they  are  not  of  a  proteid  nature.  Sidney 
Martin  has  pointed  out  that  there  is  much  that  is  analogous 
between  the  production  of  toxins  and  the  production  of  the 
bodies  of  digestion.  Just  as  ferments  are  necessary  in  the 
intestine  to  bring  about  a  change  in  the  food  by  which 


BACTERIA    AND  DISEASE  2?$ 

the  non-soluble  albumens  shall  be  made  into  soluble  pep- 
tones and  thus  become  absorbed  through  the  intestinal  wall, 
so  also  a  ferment  may  be  necessary  to  the  production  of 
toxins.  Such  ferments  have  not  as  yet  been  isolated,  but 
their  existence  in  diphtheria  and  tetanus  is,  as  we  have 
seen,  extremely  likely.  However  that  may  be,  it  is  now 
more  or  less  established  that  there  are  two  kinds  of  toxic 
bodies,  differing  from  each  other  in  their  resistance  to  heat. 
It  may  be  that  the  one  most  easily  destroyed  by  heat  is  a 
ferment  and  possibly  an  originator  of  the  other.  A  second 
division  which  has  been  suggested  for  toxic  bodies,  and  to 
which  reference  has  been  made,  is  intracellular  and  extra- 
cellular, according  to  whether  or  not  the  poison  exists 
within  or  without  the  body  of  the  bacillus. 

Lastly,  we  may  turn  to  consider  the  action  of  the  toxins 
on  the  individual  in  whose  body-fluids  they  are  formed.  It 
is  hardly  necessary  to  say  that  any  action  which  bacteria  or 
toxins  may  have  will  depend  upon  their  virulence,  in  some 
measure  upon  their  number,  and  not  a  little  upon  the  chan- 
nel of  infection  by  which  they  have  gained  entrance.  It 
could  not  be  otherwise.  If  the  virulence  is  attenuated,  or 
if  the  invasion  is  very  limited  in  numbers,  it  stands  to  reason 
that  the  pathogenic  effects  will  be  correspondingly  small  or 
absent.  The  influence  of  the  toxins  is  twofold.  In  the 
first  place  (i.)  they  act  locally  upon  the  tissues  at  the  site  of 
their  formation,  or  at  distant  points  by  absorption.  There 
is  inflammation  with  marked  cell-proliferation,  and  this  is, 
more  or  less  rapidly,  followed  by  a  specific  cell-poisoning. 
The  former  change  may  be  accompanied  by  exudation,  and 
simulate  the  early  stages  of  abscess  formation ;  the  latter  is 
the  specific  effect,  and  results,  as  in  leprosy  and  tubercle, 
in  infective  nodules.  The  site  in  some  diseases,  like  typhoid 
(intestinal  ulceration,  splenic  and  mesenteric  change)  or 
diphtheria  (membrane  in  the  throat),  may  be  definite  and 
always  the  same.  But,  on  the  other  hand,  the  site  may 


274 


BACTERIA 


depend  upon  the  point  of  entrance,  as  in  tetanus.  The 
distant  effects  of  the  toxin  are  due  to  absorption,  but  what 
controls  its  action  it  is  impossible  to  say.  We  only  know 
that  we  do  find  pathological  conditions  in  certain  organs  at 
a  distance  and  without  the  presence  of  bacteria.  We  have 
a  parallel  in  the  action  of  drugs;  for  example,  a  drug  may 
be  given  by  the  mouth  and  yet  produce  a  rash  in  some  dis- 
tant part  of  the  body.  In  the  second  place  (ii.)  toxins 
produce  toxic  symptoms.  Fever  and  many  of  the  nervous 
conditions  resulting  from  bacterial  action  must  thus  be  class- 
ified. We  have,  it  is  true,  the  chemical  symptoms  of  the 
pathological  tissue  change,  for  example,  the  large  spleen  of 
anthrax  or  the  obstruction  from  diphtheritic  membrane. 
But,  in  addition  to  these,  we  have  general  symptoms,  as 
fever,  in  which  after  death  no  tissue  change  can  be  formed. 

We  may  now  consider  briefly  some  of  the  more  important 
types  of  disease  produced  by  bacteria: 

I.  Tuberculosis.1  As  far  back  as  1794  Baillie  drew 
attention  to  the  grey  miliary  nodules  occurring  in  tuber- 
culous tissue  which  gave  rise  to  the  term  "  tubercles." 
This  preliminary  matter  was  confirmed  by  Bayle  in  1810. 

In  1834  Laennec  described  all  caseous  deposits  as  "  tuber- 
cles," insisting  upon  four  varieties: 

(1)  Miliary,  which  were  about  the  size  of  millet  seeds,  and 
in  groups; 

(2)  Crude,  miliary  tubercles  in  yellow  masses ; 

(3)  Granular,  similar  to  the  last,  but  scattered ; 

(4)  Encysted,  a  hard  mass  of  crude  tubercle  with  a  fibrous 
or  semi-cartilaginous  capsule. 

The  tubercle  possesses  in  many  cases  a  special  structure, 
and  certain  cell-forms  frequently  occur  in  it  and  give  it  a 
characteristic  appearance.  The  central  part  of  the  tubercle 

JA  detailed  study  of  tuberculosis  from  its  pathological  and  bacteriological 
aspect  will  be  found  in  La  Tuberculose  et  son  Bacille,  pt.  i.,  Straus,  Professeur 
a  la  Faculte  de  Medecine  de  Paris. 


BACTERIA   AND  DISEASE  2?$ 

usually  contains  giant  cells  with  numerous  nuclei.  The 
uninuclear  cells  are  partly  lymphoid,  partly  large  epithelial 
or  endothelial  cells ;  these  are  called  epithelioid  cells. 

It  was  not  till  1865  that  the  specific  nature  of  tuberculosis 
was  asserted  by  Villemin.  Burdon  Sanderson  (1868-69)  in 
England  confirmed  his  work,  and  it  was  extended  by  Conn- 
heim,  who  a  few  years  later  laid  down  the  principle  that  all 
is  tubercular  which  by  transference  to  properly  constituted 
animals  is  capable  of  inducing  tuberculosis,  and  nothing  is 
tubercular  unless  it  has  this  capability. 

Klebs  (1877)  and  Max  Schiller  (1880)  described  masses  of 
living  cells  or  micrococci  in  many  tuberculous  nodules  in 
the  diseased  synovial  membrane  and  in  lupus  skin.  In  1881 
Toussaint  declared  he  had  cultivated  from  the  blood  of 
tubercular  animals  and  from  tubercles  an  organism  which 
was  evidently  a  micrococcus,  and  in  the  same  year  Aufrecht 
stated  that  the  centre  of  a  tubercle  contained  small  micro- 
cocci,  diplococci,  and  some  rods.  But  it  was  not  till  the 
following  year,  1882,  that.  Koch  discovered  and  demon- 
strated beyond  question  the  specific  Bacillus  tuberculosis. 

It  is  now  held  to  be  absolutely  proved  that  the  introduc- 
tion of  the  bacillus,  or  its  spores  or  products,  is  the  one  and 
only  essential  agent  in  the  production  of  tuberculosis.  Its 
recognised  manifestations  are  as  follows : 

Tuberculosis  in  the  lungs  =  acute  or  chronic  phthisis  ; 
1  in  the  skin  =  lupus '  / 

in   the    mesenteric    glands  =  Tabes  mesen- 

terica  ; 
"  in  the  brain  =  hydrocephalus  ; 

in  lymphatic  glands  =  Scrofula.1 

The  disease  may  occur  generally  throughout  the  body  or 
locally  in  the  suprarenal  capsules,  prostate,  intestine,  larynx, 
membranes  of  the  heart,  bones,  ovaries,  pleura,  kidneys, 
spleen,  testicles,  Fallopian  tubes,  uterus,  etc. 

1  For  differences  of  virulence  between  these  conditions  of  pulmonary  tubercle 
see  Lingard,  Local  Government  Board  Report,  1888,  p.  462. 


2/6  BACTERIA 

We  may  summarise  the  history  of  the  pathology  of 
tubercle  thus : 

1794.  Baillie  drew  attention  to  grey  miliary  nodules  oc- 
curring in  tuberculosis,  and  called  them  "  tuber- 
cles." 

1834.  Laennec  described  four  varieties :  miliary;  crude; 
granular  ;  encysted. 

1843.  Klencke  produced  tuberculosis  by  intravenous  in- 
jection of  tubercular  giant  cells. 

1865.  Villemin  demonstrated  infectivity  of  tubercular 
matter  by  inoculation  of  discharges;  Connheim, 
Armanni,  Burdon  Sanderson,  Wilson  Fox,  and 
others  showed  that  nothing  but  tubercular  mat- 
ter could  produce  tuberculosis. 

1877.  Living  cells  were  found  in  tubercles,  "  micro- 
cocci  "  (Klebs,  Toussaint,  Schiller). 

1882.  Koch  isolated  and  described  the  specific  bacillus, 
and  obtained  pure  cultivations  (1884). 

The  Bacillus  of  Koch,  1882.  Delicate  cylindrical  rods, 
measuring  1.5-4  micromillimetres  in  length  and  about  .2  /i 
in  breadth;  non-motile.  Many  are  straight  with  rounded 
ends;  others  are  slightly  curved.  They  are  usually  solitary, 
but  may  occur  in  pairs,  lying  side  by  side  or  in  small  masses. 
They  are  chiefly  found  in  fresh  tubercles,  more  sparingly  in 
older  ones.  Some  lie  within  the  giant  cells;  others  lie  out- 
side ;  shorter  in  tissue  sections  of  bovine  tuberculosis,  but 
longer  in  the  milk  (Crookshank). 

When  stained  they  appear  to  be  composed  of  irregular 
cubical  or  spherical  granules  within  a  faintly  stained  sheath. 
In  recent  lesions  the  protoplasm  appears  more  homogeneous, 
and  takes  on  the  segmented  or  beaded  character  only  in  old 
lesions,  pus,  or  sputum. 


BACTERIA    AND  DISEASE  277 

Morphological  differences  are  found  under  different  cir- 
cumstances, and  within  limits  variation  occurs  according  to 
the  environment. 

Cultivation  on  Various  Media.  Koch  inoculated  solid  blood 
serum  with  tubercular  matter  from  an  infected  lymphatic 
gland  of  a  guinea-pig,  and  noticed  the  first  signs  of  growth 
in  ten  or  twelve  days  in  the  form  of  whitish,  scaly  patches. 
These  enlarged  and  coalesced  with  neighbouring  patches, 
forming  white,  roughened,  irregular  masses.  Nocard  and 
Roux  showed  that  by  adding  f  per  cent,  of  glycerine  to  the 
media  commonly  used  in  the  laboratory,  such  as  nutrient 
agar  or  broth,  the  best  growth  is  obtained. 

On  glycerine  broth  or  glycerine  agar  abundant  growth  ap- 
pears at  the  end  of  seven  or  eight  days.  By  continuous 
sub-culture  on  glycerine  agar  the  virulence  of  the  bacillus  is 
diminished.  But  in  fifteen  days  after  inoculation  of  the 
medium  the  culture  equals  in  extent  a  culture  of  several 
weeks'  age  on  blood  serum. 

Sub-cultures  from  glycerined  media  will  grow  in  ordinary 
broth  without  glycerine  (Nocard,  Roux,  Crookshank). 

In  alkaline  broth  to  which  a  piece  of  boiled  white  of  egg 
was  added  Klein  obtained  copious  growths,  and  found  that 
continued  sub-culturing  upon  this  medium  also  lessens  the 
virulence. 

Description  of  Cultivations: — On  glycerine  agar  minute 
white  colonies  appear  in  about  six  days,  raised  and  isolated, 
and  coalescing  as  time  advances,  forming  a  white  lichenous 
growth,  fully  developed  in  about  two  months. 

On  glycerine  broth  a  copious  film  appears  on  the  surface 
of  the  liquid,  which  if  disturbed  falls  to  the  bottom  of  the 
flask  as  a  deposit. 

Spore  Formation.  In  very  old  cultivations  spore-like 
bodies  can  be  observed  both  in  stained  and  unstained  pre- 
parations, but  neither  the  irregular  granules  within  the  cap- 
sule nor  the  unstained  spaces  between  the  granules  are 


2/8  BACTERIA 

spores  (Babes  and  Crookshank).     That  the  bacilli  possess 
spores  is  believed  on  account  of  the  following  facts: 

1.  That  tubercular  sputum,  when  thoroughly  dried,  main- 
tains its  virulent  character  (Koch,  Schill,  Fischer,  etc.).     No 
sporeless   bacillus   is   known    which    can    survive   through 
drying. 

2.  That  tubercular  matter  and  cultures  survive  temper- 
ature up  to  100°  C.     Non-spore-bearing  bacilli  and  micro- 
cocci  are  killed  by   being  exposed   for  five  minutes  to  a 
temperature  of  65-70°  C.,  whereas  spores  of  other  bacilli 
withstand  much  higher  temperatures. 

3.  Tubercular   sputum  distributed  in  salt  solution  does 
not  lose  its  virulence  by  being  kept  at   100°  C.  for  one  or 
two    minutes;    sporeless    bacilli    certainly    would    (Klein). 

4.  A  solution  of  per-chloride  of  mercury  does  not  kill  the 
tubercle  bacilli,   as  it  does  sporeless  bacilli  (Lingard  and 
Klein). 

Koch  and  many  bacteriologists  have  declared  the  bacillus 
to  be  a  "  true  parasite."  Koch  based  this  view  upon  the 
belief  which  he  entertained  that  the  bacillus  can  grow  only 
between  30°  C.  and  41°  C.,  and  therefore  in  temperate 
zones  is  limited  to  the  animal  body  and  can  originate  only 
in  an  animal  organism.  "  They  are,"  he  said,  "  true  para- 
sites, which  cannot  live  without  their  hosts.  They  pass 
through  the  whole  cycle  of  their  existence  in  the  body." 
But  at  length  Koch  and  others  overcame  the  difficulties  and 
grew  the  bacillus  as  a  saprophyte. 

Schottelius  l  has  observed  that  tubercle  bacilli  taken  from 
the  lung  of  phthisical  persons  buried  for  years  still  retains 
its  virulence  and  capability  of  producing  tuberculosis  upon 
inoculation.  He  further  shows  that  tubercular  lung  kept 
in  soil  (enclosed  in  a  box)  shows  a  marked  rise  in  temper- 
ature. Klein  quotes  these  experiments  as  indications  that 

tubercle  bacilli  are  not  true  parasites,  but  belong  to  the  ecto- 

1  Centralblatt.  f.  Bact.  und  Parasit.,  vol.  vii.,  p.  9. 


BACTERIA   AND  DISEASE  279 

genie  microbes  which  can  live  and  thrive  independent  of  a 
living  host. ' ' 

It  has  now  been  abundantly  proved  that  the  bacillus  of 
tuberculosis  is  capable  of  accommodating  itself  to  circum- 
stances much  less  favourable  than  had  been  supposed, 
especially  as  regards  temperature. 

Temperature  of  Growth  of  Bacillus.  30-41°  C.  have  been 
laid  down  by  Koch  as  the  limits  of  temperature  at  which  the 
bacillus  will  grow  in  culture  medium  outside  the  body.  The 
generally  accepted  temperatures  as  most  favourable  to 
the  growth  of  the  bacillus  are  between  36°  C.  and  38°  C. 

Sir  Hugh  Beevor,  however,  was  able  to  grow  the  bacillus 
upon  glycerine  agar  at  28°  C.  (82°  F.),  obtaining  an  ample 
culture  which  developed  somewhat  more  slowly  than  on 
blood  serum,  and  to  a  less  extent  than  at  37°  C.  In  both 
Beevor  succeeded  in  growing  the  bacillus  at  a  lower  temper- 
ature even  than  on  agar,  viz.,  at  a  temperature  rarely  above 
60°  F.  Sheridan  Delepine  and  others  have  also  been  suc- 
cessful in  obtaining  growths  at  room  temperature  both  in 
summer  and  winter. 

Although,  speaking  generally,  there  is  an  actual  cessation 
of  growth  at  low  temperature,  the  bacillus  may  be  exposed 
to  very  low  temperatures  for  a  considerable  time  without 
losing  its  power  of  again  becoming  active  when  returned  to 
a  favourable  environment  (Woodhead). 

The  Relation  of  the  Bacillus  to  the  Disease.  All  four  of 
Koch's  postulates  have  been  fulfilled  in  the  case  of  Bacillus 
tuberculosis.  Hence  we  are  dealing  with  the  specific  cause 
of  the  disease.  Yet,  whilst  this  is  so,  we  may  usefully  ask 
ourselves:  How  does  the  bacillus  set  up  the  changes  in 
normal  tissues  which  result  in  tubercular  nodules  ?  In 
arriving  at  a  solution  of  this  problem  we  are  materially  aided 
if  we  bear  in  mind  the  fact  that  such  an  organism  in  healthy 
tissues  has  a  double  effect.  First,  there  is  an  ordinary  in- 
flammatory irritation,  and  secondly,  there  is  a  specific  change 


280  BACTERIA 

set  up  by  the  toxins  of  the  bacillus.  Directly  the  invading 
bacilli  find  themselves  in  a  favourable  nidus  they  commence 
multiplication.  In  three  or  four  days  this  acts  as  an  irritant 
upon  the  surrounding  connective-tissue  cells,  which  prolifer- 
ate, and  become  changed  into  large  cells  known  as  epithelioid 
cells.  At  the  periphery  of  this  collection  of  epithelioid  cells 
we  have  a  congested  area.  This  change  has  been  accom- 
plished by  the  presence  of  the  bacilli  themselves.  The  pro- 
duction of  their  specific  poisons  changes  the  epithelioid  cells 
in  the  centre  of  the  nodule,  some  of  which  become  fused 
together,  whilst  others  expand  and  undergo  division  of 
nucleus.  By  this  means  we  obtain  a  series  of  large  multi- 
nucleated  cells  named  giant  cells.  If  the  disease  is  very 
active,  these  soon  caseate  and  break  down  in  the  centre. 
In  a  limb  we  get  a  discharge ;  in  a  lung  we  get  an  expector- 
ation. Both  discharge  and  expectoration  arise  from  a  break- 
ing down  of  the  new  cell  formation.  Previously  to  breaking 
down  we  have  in  a  fully  developed  nodule  healthy  tissue, 
inflammatory  zone,  epithelioid  cells,  giant  cells,  containing 
nuclei  and  bacilli.  The  sputum  or  the  discharge  will,  dur- 
ing the  acute  stage  of  the  disease  at  all  events,  contain 
countless  numbers  of  the  bacilli,  which  may  thus  be  readily 
detected,  and  their  presence  used  as  evidence  of  the  disease. 
It  is  obvious  that  if  the  centre  of  the  nodule  degenerates 
and  comes  away  as  discharge  a  cavity  will  be  left  behind. 
By  degrees  this  small  cavity  may  become  a  very  large  one, 
as  is  frequently  the  case  in  the  lung,  which  particularly 
lends  itself  to  such  a  condition.  Hence,  though  at  the  out- 
set a  tubercular  lung  is  solid,  at  the  end  it  is  hollow. 

The  exact  period  of  giant-cell  formation  depends  on  the 
rapidity  of  the  formative  processes.  Thus  different  condi- 
tions occur.  Inside  the  giant  cells  the  bacilli  are  arranged 
in  relation  to  the  nuclei  in  one  of  three  ways:  (a)  polar,  (b) 
zonal,  or  (c)  mixed.  The  breaking  down  of  the  nodule  is 
partly  due  to  the  cell-poisons,  and  partly  because  the  nodule 


S       V 


V- 


1 

' 


BACILLUS  TUBERCULOSIS 

(In  sputum  from  a  case  of  phthisis,  "consump- 
tion "  of  the  lungs) 
X   1000 

By  permission  of  the  Scientific  Press,  Limited 


BACILLUS  TUBERCULOSIS 

(The  bacilli  are  arranged  within  the  giant  cell) 
X  1000 


STREPTOCOCCUS  PYOGENES 

(From  broth  culture) 
X  looo 


BACILLUS  ANTHRACIS 

(From  splenic  blood  of  cow) 
X  looo 


By  permission  of  the  Scientific  Press,  Limited  By  permission  of  the  Scientific  Press,  Limited 


BACTERIA    AND   DISEASE  28 1 

is  non-vascular,  owing  to  the  fact  that  new  capillaries  can- 
not grow  into  the  dense  nodule,  and  the  old  ones  are  all 
occluded  by  the  growth  of  the  nodule. 

From  the  local  foci  of  disease  the  tubercle  process  spreads 
chiefly  by  three  channels : 

(a)  By  the  lymphatics,   affecting  particularly  the  glands. 
Thus  we  get  tuberculosis  set  up  in  the  bronchial,  tracheal, 
mediastinal,  and  mesenteric  glands,  and  it  is  so  frequently 
present  as  to  be  a  characteristic  of  the  disease.     This  is  the 
common  method  of  dissemination  in  the  body. 

(b)  By  the  blood-vessels,  by  means  of  which  bacilli  may  be 
carried  to  distant  organs. 

\c]  By  continuity  of  tissues,  infective  giant-cell  systems 
encroaching  upon  neighbouring  tissues,  or  discharge  from 
lungs  or  bronchial  glands  obtaining  entrance  to  the  gullet 
and  thus  setting  up  intestinal  disease  also. 

It  has  been  abundantly  proved  that  the  respiratory  and 
digestive  systems  are  principally  affected  by  Koch's  bacillus. 
Wherever  the  bacilli  are  arrested,  they  excite  formation  of 
granulations  or  miliafy  tubercular  nodules,  which  increase 
and  eventually  coalesce.  The  lymphatic  glands  which  col- 
lect the  lymph  from  the  affected  region  are  the  earliest 
affected,  always  the  nearest  first,  and  then  the  disease  ap- 
pears to  be  appreciably  stopped  on  its  invading  march. 
Each  lymphatic  gland  acts  as  a  temporary  barrier  to  pro- 
gress until  the  disease  has  broken  its  structure  down.  It 
remains  local,  in  spite  of  increase  in  number  and  importance 
of  the  foci  of  disease,  as  long  as  the  bacilli  have  not  gained 
access  to  the  blood  stream. 

Toxins  and  Tuberculin.  Koch,  Crookshank,  and  Herroun, 
Hunter,  and  others  have  isolated  products  from  pure  cult- 
ures of  the  tubercle  bacillus.  These  have  comprised  chiefly 
albumoses,  alkaloids,  and  various  extractives.  Koch's  ob- 
servations led  him  to  suppose  that  in  pure  cultures  of  tubercle 
a  substance  appeared  having  healing  action  on  tuberculosis, 


282  BACTERIA 

and  an  extract  of  this  in  glycerine  he  termed  "  tuberculin." 
It  was  made  as  follows :  A  veal  broth  containing  peptone 
and  glycerine  was  inoculated  with  a  pure  culture  of  the 
bacillus  and  incubated  at  38°  C.  for  six  or  eight  weeks.  An 
abundant  growth  with  copious  film  formation  appeared. 


FLASK  USED  IN  THE  PREPARATION  OF  TUBERCULIN 

The  culture  was  then  concentrated  by  evaporation  over  a 
water-bath  until  reduced  to  about  one-tenth  of  its  volume. 

The  announcements  in  1890  and  1891  to  the  effect  that  a 
"  cure  "  had  been  discovered  for  consumption  will  be  re- 
membered. The  hopes  thus  raised  were  unfortunately  not 
to  be  realised.  Koch  advocated  injections  of  this  tuberculin 
in  cases  of  skin  tubercle  (lupus)  and  consumptive  cases.  In 
many  of  these  benefit  was  apparently  derived,  but  its  gen- 
eral application  was  not  founded  upon  any  substantial  basis. 
Dead  tissue,  full  of  bacilli,  could  not  thus  be  got  rid  of;  nor 
could  the  career  of  the  isolated  bacilli  distributed  through 
the  body  be  thus  checked. 

Tuberculin  has,  however,  found  a  remarkable  sphere  of 
usefulness  in  causing  reaction  in  animals  suffering  from 
tuberculosis.  Indeed,  tuberculin  is  the  most  valuable  means 
of  diagnosis  that  we  possess  (MacFadyen).  When  injected 
(dose,  30-40  centigrammes)  it  causes  a  rise  of  one  and  a  half 
to  three  degrees.  The  fever  begins  between  the  twelfth  and 
fifteenth  hour  after  injection,  and  lasts  several  hours.  The 
duration  and  intensity  of  the  reaction  have  no  relation  to 
the  number  and  gravity  of  the  lesions,  but  the  same  dose 
injected  into  healthy  cattle  causes  no  appreciable  febrile 
reaction.  The  tuberculous  calf  reacts  just  as  well  as  the 


BACTERIA   AND  DISEASE  283 


adult,  but  the  dose  is  generally  10-20  centigrammes.  In- 
jections of  tuberculin  have  no  troublesome  effect  on  the 
quantity  or  quality  of  the  milk  of  cows  or  on  the  progress 
of  gestation. 

Tuberculosis  of  Animals.  Cattle  come  first  amongst 
animals  liable  to  tubercle.  Horses  may  be  infected,  but  it 
is  comparatively  rare,  and  among  small  ruminants  the  dis- 
ease is  rarer  still.  Dogs,  cats,  and  kittens  may  be  easily 
infected.  Amongst  birds,  fowls,  pigeons,  turkeys,  and 
pheasants,  the  disease  assumes  almost  an  epidemic  character. 
Especially  do  animals  in  confinement  die  of  tubercle,  as  is 
illustrated  in  zoological  gardens.  Respecting  the  lesions  of 
bovine  tuberculosis,  it  will  be  sufficient  to  say  that  nothing 
is  more  variable  than  the  localisation  or  form  of  its  attacks. 
The  lungs  and  lymphatic  glands  come  first  in  order  of  fre- 
quency, next  the  serous  membranes,  then  the  liver  and 
intestines,  and  lastly  the  spleen,  joints,  and  udder  (Nocard). 

The  anatomical  changes  in  bovine  tubercle  are  mostly 
found  in  the  lungs  and  their  membranes,  the  pleurae.  It 
also  affects  the  internal  membrane  lining,  the  abdomen  and 
its  chief  organs,  the  peritoneum,  and  the  lymphatic  glands. 
In  both  these  localities  a  characteristic  condition  is  set  up 
by  small  grey  nodules  appearing,  which  increase  in  size, 
giving  an  appearance  of  "  grapes."  Hence  the  condition 
is  called  grape  disease,  or  Perlsucht.  The  organs,  as  we  have 
said,  are  equally  affected,  and  when  we  add  the  lymphatic 
glands  we  have  a  fairly  complete  summary  of  the  form  of 
the  disease  as  it  occurs  in  cattle.  As  has  been  clearly 
pointed  out  by  Martin,  Woodhead,  and  others  in  their  evi- 
dence before  the  Royal  Commission,  the  organs,  glands, 
and  membranes  are  the  sites  for  tubercle,  not  the  muscles 
(or  "  meat  ").  This  latter  is  most  liable  to  convey  infection 
when  the  butcher  smears  it  with  the  knife  which  he  has 
used  to  remove  tubercular  organs. 

As  regards  the  udder  in  its  relation  to  milk  infection,  it 


284  BACTERIA 

may  be  desirable  to  state  that  the  initial  lesion,  according 
to  Nocard  and  Bang,  takes  the  form  of  a  progressive  scler- 
osis. The  interlobular  connective  tissue,  normally  scanty, 
becomes  thickened,  fibrous,  and  infiltrated  by  minute  mili- 
ary  granulations.  The  granular  tissue  is  thus  "  smothered 
by  the  hypertrophy  and  fibrous  transformation  of  the  inter- 
stitial connective  tissue  "  (Nocard).  The  walls  of  the  ducts 
are  thickened  and  infiltrated,  the  lumen  frequently  dilated 
by  masses  of  yellow  caseous  material.  On  the  whole  it  may 
be  said  that  tubercle  of  the  udder  is  rare.  Usually  only 
one  quarter  is  attacked,  and  by  preference  the  posterior. 
For  some  time  the  milk  remains  normal,  but  gradually  it 
becomes  serous  and  yellow,  and  contains  coagula  holding 
numbers  of  bacilli.  Lastly,  it  becomes  purulent  and  dries 
up  altogether.  While  the  milk  is  undergoing  these  changes 
the  lesion  of  the  udder  is  becoming  more  marked,  the  tissue 
becomes  less  supple,  and  the  toughness  increases  almost  to 
a  wooden  hardness. 

The  general  anatomical  characteristics  of  the  disease  are 
similar  to  those  occurring  in  man. 

The  percentage  of  cattle  suffering  from  tubercle  varies. 
In  Germany  it  appears  to  vary  from  2  to  8  per  cent,  of  all 
cattle,  in  Saxony  17  to  30  per  cent.,  in  England  22  per  cent, 
approximately  (in  London  40  per  cent.),  in  France  25  per 
cent.  Lowland  breeds  are  much  more  infected  than  moun- 
tain breeds,  which  possess  stronger  constitutions. 

Tuberculosis  of  the//^  is  less  common  than  that  of  cattle, 
but  not  so  rare  as  that  of  the  calf  (Nocard).  In  nine  out  of 
ten  cases  the  pig  is  infected  by  ingestion,  particularly  when 
fed  on  the  refuse  from  dairies  and  cheese  factories.  The 
disease  follows  the  same  course  as  in  cattle.  The  finding  of 
the  bacillus  is  difficult,  and  the  only  safe  test  is  inoculation 
(Woodhead). 

Sheep  are  very  rarely  tuberculous  by  nature,  though  there 
is  evidence  to  believe  that  very  long  cohabitation  with 


BACTERIA    AND  DISEASE  285 

tuberculous  cattle  would  succeed  in  transmitting  tubercu- 
losis to  some  sheep. 

Tuberculosis  in  the  horse  is  relatively  very  rare.  It  attacks 
the  organs  of  the  abdominal  cavity,  especially  the  glands; 
it  affects  the  lung  secondarily  as  a  rule.  The  cases  are  gen- 
erally isolated  ones,  even  though  the  animal  belongs  to  a 
stud.  Nocard  holds  that  the  bacillus  obtained  from  the 
pulmonary  variety  is  like  the  human  type,  whilst  the  ab- 
dominal variety  is  more  like  the  avian  bacillus. 

Nocard  says  1  : 

"  If  the  dog  can  become  tuberculous  from  contact  with  man, 
the  converse  is  equally  true.  Infection  is  at  any  rate  possible 
when  a  house-dog  scatters  on  the  floor,  carpet,  or  bed,  during 
its  fit  of  coughing,  virulent  material,  which  is  rendered  extremely 
dangerous  by  drying,  especially  for  children,  its  habitual  play- 
mates. The  most  elementary  prudence  would  recommend  the 
banishment  from  a  room  of  every  dog  which  coughs  frequently, 
even  though  it  only  seems  to  be  suffering  from  some  common* 
affection  of  the  bronchi  or  lung." 

Tuberculosis  is  a  common  disease  among  the  birds  of  the 
poultry-yard:  poultry,  pigeons,  turkeys,  pea-fowl,  guinea- 
fowl,  etc.  They  are  infected  almost  exclusively  through 
the  digestive  tract,  generally  by  devouring  infected  secre- 
tions of  previous  tubercular  fowls,  Whatever  the  position 
or  form  of  avian  tuberculosis,  the  bacilli  are  present  in 
enormous  numbers,  and  are  often  much  shorter  and  some- 
times much  longer  than  those  met  with  in  tuberculous 
mammalia,  and  grow  outside  the  body  at  a  higher  temper- 
ature (43°  C.).  They  are  also  said  to  be  more  resistant  and 
of  quicker  growth.  The  species  is  probably  identical  with 
Koch's  bacillus,  though  there  are  differences.  In  the  nodule, 
which  is  larger  than  in  human  tuberculosis,  there  are  few  or 
no  giant  cells,  and  it  does  not  so  readily  break  down. 

1  Animal  Tuberculosis,  p.  129. 


286  BACTERIA 

Nocard  and  others  have  demonstrated  the  fact  that  the 
Bacillus  tuberculosis  of  Koch  is  the  common  denominator  in 
all  tubercular  disease,  whatever  and  wherever  its  manifest- 
ations, in  all  animals.  The  bacillus,  they  hold,  may, 
however,  experience  profound  modifications  by  means  of 
successive  passages  through  the  bodies  of  divers  species  of 
animals.  But  if  the  modifications  which  it  undergoes  as  a 
result  of  transmissions  through  birds,  for  example,  are  pro- 
found enough  to  make  the  bacillus  of  avian  tubercle  a 
peculiar  variety  of  Koch's  bacillus,  they  are  not  enough,  it  is 
generally  believed,  to  make  these  bacilli  two  distinct  species. 

We  may,  therefore,  take  it  for  granted  that  tuberculosis 
is  one  and  the  same  disease,  with  various  manifestations, 
common  to  man  and  animals,  intercommunicable,  and  hav- 
ing but  one  vera  causa  :  the  Bacillus  tuberculosis  of  Koch. 

The  Prevention  of  Tuberculosis.  At  the  present  time 
much  attention  is  being  directed  to  the  administrative  per- 
sonal control  of  tuberculosis.  How  greatly  this  is  needed 
in  so  preventable  a  disease  is  evident  from  a  perusal  of  the 
following  quotation  from  the  Registrar-General's  reports. 
(See  opposite  page.) 

These  figures  show  a  marked  decline  in  the  three  worst 
forms  of  the  disease.  But  this  decline  is  apparently  less 
marked  in  tabes  than  in  phthisis  or  tubercular  meningitis, 
i.  e.,  less  in  the  kind  of  tubercle  due  to  the  ingestion  of 
infected  milk.  Fortunately  the  State  is  beginning  to  realise 
its  duty  in  regard  to  preventive  measures.  The  abolition 
of  private  slaughter-houses,  the  protection  of  meat  and  milk 
supplies,  the  seizure  of  tuberculous  milch  cows,  and  such 
like  measures  fall  obviously  within  the  jurisdiction  of  the 
State  rather  than  the  individual,  and  claim  the  earnest  and 
urgent  attention  of  the  public  health  departments  of  states.1 

1  See  the  Harben  Lectures,  November,  1898,  by  Sir  Richard  Thome  Thorne, 
Medical  Officer  to  the  Local  Government  Board  ;  also  the  Report  of  the  Royal 
Commission  on  Tuberculosis,  1896-98. 


BACTERIA   AND   DISEASE 


287 


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288  BACTERIA 

But  personal  hygiene  and  the  prevention  of  the  transmission 
of  the  disease  depend  very  largely  indeed  upon  the  mass  of 
the  population.  Hence  we  hail  with  satisfaction  the  recent 
endeavours  to  educate  public  opinion.  In  order  to  make 
this  matter  very  simple  indeed,  we  have  placed  in  a  footnote 
a  series  of  statements  embodying  some  of  the  chief  facts 
which  every  individual  in  our  crowded  communities  should 
know.1 

1  i.  Tuberculosis  is  a  disease  mainly  affecting  the  lungs  (consumption,  decline, 
phthisis)  of  young  adults  and  the  bowels  of  infants  (tabes  mesentericd).  It  may 
affect  any  part  of  the  body,  and  its  manifestations  are  very  various.  It  also 
affects  animals,  particularly  cattle,  by  whom  it  may  be  transmitted  to  man. 

2.  Its  direct  cause  is  a  microscopic  vegetable  cell,  known  as  the  Bacilhis 
tuberculosis,  discovered  by  Koch  in  1882.     This  fungus  requires  to  be  magni- 
fied some  hundreds  of  times  before  it  can  even  be  seen.    When  it  gains  entrance 
to  the  weakened  body  it  sets  up  the  disease,  which  is  an  infectious  disease, 
though  different  in  degree  to  the  infectiousness  of,  say,  measles. 

3.  Trade  influence  and  occupation,  in  some  cases,  undoubtedly  predispose 
the  individual  to  tubercle.     Cramped  attitudes,  exposure  to  dampness  or  cold, 
ill  ventilation,  and  exposure  to  inhalation  of  dust  of  various  kinds,  all  act  in 
this  way.     In  support  of  the  evil  effect  of  each  of  these  four  conditions  much 
evidence  could  be  produced. 

4.  Overcrowding  has  a  definite  influence  in  propagating  tubercular  diseases. 
The  agricultural  counties  without  big  towns,  like  Worcestershire,  Hereford- 
shire, Buckinghamshire,  and  Rutland,  are  the  counties  having  the  lowest  mor- 
tality from  tuberculosis  ;  whilst  the  crowded  populations  in  Northumberland, 
South  Wales,  Lancashire,  London,  and  the  West  Riding  suffer  most.    Speaking 
more   particularly,    the    overcrowded    areas    of    London,   such   as    St.    Giles', 
Strand,   Holborn,  and  Central   London   generally,  show  very  high  tubercular 
death-rates. 

5.  Tuberculosis  is  not  increasing.     During  the  last  thirty  years  it  has  shown, 
with  few  exceptions,  a  steady  decline  in  all  parts  of  England.    "  Consumption  " 
is  most  fatal  in  comparatively  young  people  (fifteen  to  forty-five  years),  whilst 
"  tabes  "  and  other  forms  of  tubercle  are  fatal  chiefly  to  young  children.     These 
forms  have  not  declined  so  much  as  the  lung  form.     The  mortality  in  con- 
sumption of  males  has  since  1866  been  in  excess  of  that  of  females.     The  age 
of  maximum  fatality  from  consumption  is  later    than  in  the  past,  which  is 
probably  due  to  improved  hygiene  and  treatment. 

6.  This  decline  has  been  due,  not  to  any  special  repressive  measures — for  few 
or  none  have  been  carried  out — but  to  a  general  and  extensive  social  improve- 
ment in  the  life  of  the  people,  to  an  increase  of  knowledge  respecting  tubercu- 


BACTERIA   AND  DISEASE  289 

Diphtheria  (Klebs-Loffler  Bacillus,  1882-1884).  Diph- 
theria is  an  infective  disease  characterised  by  a  variety  of 
clinical  symptoms,  but  commonly  by  a  severe  inflammation 
followed  by  a  fibrous  infiltration  (constituting  a  membrane) 
of  certain  parts.  The  membrane  ultimately  breaks  down. 


BACILLUS  OF  DIPHTHERIA 

losis  and  hygiene,  to  an  enormous  advance  in  sanitation,  and  to  more  efficient 
land  drainage. 

7.  Not  all  persons  are  equally  liable  to  consumption,  some  being  much  more 
susceptible  than  others.      We  have  mentioned  the  predisposing  influence  of 
certain  trades.     There  is  also  heredity,  which  acts,  as  we  have  said,  in  trans- 
mitting a  tubercular  tendency,  not  commonly  the  actual  virus  of  the  disease  ; 
there  is,  thirdly,  the  debilitating  effect  of  previous  illness  or  chronic  alcohol- 
ism ;  there  is,  fourthly,  the  habitual  breathing  of  rebreathed  air  ;  and,  fifthly, 
there  are  the  conditions  of  the  environment,  like  dampness  and  darkness  of  the 
dwelling.     Such  influences  as  these  weaken  the  resisting  power  of  the  tissues, 
and  thus  afford  a  suitable  nidus  for  the  bacillus  conveyed  in  milk  or  by  the 
inspiration  of  infected  dust. 

8.  Consumption  is  curable  if  taken  in  time.     In  cases  where  the  lungs  are 
half  gone,  and  consist  of  large  cavities,  it  is  obvious  that  curability  is  out  of 
the  question.     But  if  the  disease  can  be  properly  treated  in  its  earliest  stages, 
there  is  considerable  likelihood  of  recovery. 

9.  The  breath  is  not  dangerous,  as  far  as  we  know,  but  there  is  danger  from 
discharges  of  any  kind  from  any  infected  part,  whether  lungs  or  bowels  ;  for 
such  discharges,  when  dry,  may  readily  pollute  the  air,  and  either  the  bacilli 
or  spores  be  inhaled  into  the  lungs. 

10.  The  chief  channels  of  personal  infection  or  the  spread  of  the  disease 
amongst  a  community  are  two  :  (a)  dried  tubercular  sputum  (or  other  tubercu- 
lar discharges) ;  (b)  infected  milk  or  meat.     So  long  as  the  former  remains  wet 

19 


BACTERIA 

The  parts  affected  are  the  mucous  membrane  of  the  fauces, 
larynx,  pharynx,  trachea,  and  sometimes  wounds  and  the 
inner  wall  of  the  stomach.  The  common  sign  of  the  disease 
is  the  membrane  in  the  throat;  but  muscle  weakness, 
syncope,  albuminuria,  post-diphtheritic  paralysis,  convul- 
sions, and  many  other  symptoms  guide  the  physician  in 
diagnosis  and  the  course  of  the  disease. 

The  Bacillus  diphtheria  was  isolated  from  the  many  bac- 
teria found  in  the  membrane  by  Loffler.  Klebs  had  pre- 
viously identified  the  bacillus  as  the  cause  of  the  disease. 

or  moist,  infection  cannot  take  place.  It  is,  of  course,  better  to  destroy  it 
completely.  As  for  milk  and  meat,  boiling  the  former  and  thoroughly  cook- 
ing the  latter  will  remove  all  danger. 

11.  The  expectoration  is  infective.     This  is  one  of  the  commonest  modes  of 
infection,  and  to  it  is  held  to  be  due  the  large  amount  of  respiratory  tubercu- 
losis (consumption,  phthisis).     The  expectoration  from  the  lungs  must  contain, 
from  the  nature  of  the  case,  a  very  large  number  of  bacilli.     As  a  matter  of 
fact,  a  single  consumptive  individual  can  cough  up  in  a  day  millions  of  tuber- 
cle bacilli.     When  expectoration    becomes  dry,   the  least  current  of  air  will 
disseminate  the  infective  dust,  which  can  by  that  means  be  readily  reinspirecl. 
Expectoration  on  pavements  and  floors,  as  well  as  on  handkerchiefs,  may  thus  be- 
come, on  drying,  a  source  of  great  danger  to  others.     The  discharges  from  the 
bowels  of  infants  suffering  from  the  disease  also  contain  the  infective  material. 

12.  Milk,  though  a  much  more  likely  channel  for  conveyance  of  tubercle 
than  meat,  is  only  or  chiefly  virulent  when  the  udder  is  the  seat  of  tuberculous 
lesions.     The  consumption  of  such  milk  is  only  dangerous  when  it  contains  a 
great  number  of  bacilli  and  is  ingested  in  considerable  quantity.     Practically 
the  danger  from  using  raw  milk  exists  only  for  those  persons  who  use  it  as 
their  sole  or  principal  food,  e.  g.,  young  children.     All  danger  is  avoided  by 
boiling  or  pasteurising  the  milk. 

At  the  same  time  there  is  an  increasing  amount  of  evidence  forthcoming  at 
the  present  time  which  goes  to  prove  that  milk  is  not  infrequently  tainted  with 
tubercle  (see  p.  195).  The  tuberculin  test  should  be  applied  to  all  milch  cows, 
and  the  infected  ones  isolated  from  the  herd.  Milk  supplies  should  be  more 
strictly  inspected  even  than  cowsheds. 

13.  There  are  several  methods  by  which  meat  infection  can  be  prevented.    In 
the  first  place,  herds  should  be  kept  healthy,  and  tubercular  animals  isolated. 
Cowsheds   and    byres   should    be   under   sanitary   supervision,    especially   as 
regards  overcrowding,  dampness,   lack  of   light,  and  uncleanliness.      Public 
slaughter-houses  under  a  sanitary  authority  would  undoubtedly  be  most  ad- 


BACTERIA   AND  DISEASE  29! 

It  is  a  slender  rod,  straight  or  slightly  curved,  and  remark- 
able for  its  beaded  appearance ;  there  are  also  irregular  and 
club-shaped  forms.  It  differs  in  size  according  to  its  culture 
medium,  but  is  generally  3  or  4  /u,  in  length.  In  the  mem- 
brane which  is  its  strictly  local  habitat  in  the  body — indeed, 
the  bacillus  is  found  nowhere  else  in  the  body — it  almost 
invariably  shows  parallel  grouping,  lying  between  the  fibrin 
of  the  membrane,  and  most  largely  in  its  deeper  parts. 
Here  it  is  mixed  with  other  bacilli,  micrococci,  staphylococci, 
and  streptococci,  all  of  which  are  present  and  performing 

vantageous.  Meat  inspection  should  also  be  more  strictly  attended  to  ;  efficient 
cooking,  and  avoidance  of  "  roll "  meat  which  has  not  been  thoroughly  cooked 
in  the  middle. 

14.  Consumptive  patients  may  diminish  their  disease.     Dr.  Arthur  Ransome  l 
has  laid  down  five  axioms  of  hygiene  for  phthisical  patients  which,  if  followed, 
would  materially  improve  the  condition  of  such  persons.     At  Davos,  St.  Moritz, 
Nordrach,   and  other  places  where  they  have   been  practised,  the  beneficial 
change  has  been  in  many  cases  extraordinary  : 

(1)  Abundance  of  light,   nutritious,    easily  digested  food,  which  must 

comprise  a  large  allowance  of  fat ;  small  meals,  but  frequent ; 

(2)  An  almost  entirely  open-air  life,  with  as  much  sunshine  as  can  be 

obtained  ; 

(3)  Suitable  clothing,  mostly  wool ; 

(4)  Cleanliness  and  bracing  cold-water  treatment ; 

(5)  Mild  but  regular  exercise. 

15.  Consumptive  patients  may  also  assist  in  preventing  the  spread  of  the 
disease.     In  the  first  place,   they  should  follow  the  hygienic  directions  just 
mentioned,  because  such  conditions  fulfilled  will   materially  lessen  the  con- 
tagiousness of  such  patients  ;  next,  the  expectoration  must  never  be  allowed  to 
get  dry.     A  spitting-cup  containing  a  little  disinfectant  solution  (one  teaspoon- 
ful  of  strong  carbolic  acid  to  two  tablespoonfuls  of  water)  should  always  be 
used,   or  the  expectoration  received  into  paper  handkerchiefs  which  can  be 
burnt.     Spoons,  forks,  cups,  and  all  such  articles  should  be  thoroughly  cleaned 
before  being  used  by  other  persons.     The  patient  should  not  sleep  in  company 
with  another,  but  occupy,  if  possible,  a  separate  bedroom. 

Isolation  hospitals  for  consumptives,  as  for  patients  suffering  from  diphtheria, 
are  now  being  established. 

16.  House  influence  has  some  effect,  both  directly  and  indirectly,  upon  tuber- 
cular diseases.     Damp  soils,  darkness,  and  small  cubic  space  in  the  dwelling- 

1  Arthur  Ransome,  M.D.,  F.R.S.,  Treatment  of  Phthisis. 


2Q2  BACTERIA 

their  part  in  complicating  the  disease.  The  bacillus  pos- 
sesses five  negative  characters;  namely,  it  has  no  spores, 
threads,  or  power  of  mobility,  and  does  not  produce  lique- 
faction or  gas.  It  stains  with  Loffler's  methylene  blue,  and 
shows  metachromatic  granules  and  polar  staining.  Its 
favourable  temperature  is  blood-heat,  though  it  will  grow  at 
room  temperature.  It  is  aerobic,  and,  indeed,  prefers  a 
current  of  air.  Loffler  contrived  a  medium  for  cultivation 
which  has  proved  most  successful.  It  is  made  by  mixing 
three  parts  of  ox-blood  serum  with  one  part  of  broth  con- 
taining I  per  cent,  of  glucose,  I  per  cent,  of  peptone,  and  ^ 
per  cent,  of  common  salt ;  the  whole  is  coagulated.  Upon 
this  medium  the  Klebs-Loffler  bacillus  grows  rapidly  in 
eighteen  or  twenty  hours,  producing  scattered  "  nucleated  " 
round  white  colonies,  becoming  yellowish.  It  grows  well 
in  broth,  but  without  producing  either  a  pellicle  or  turbidity  ; 
it  can  grow  on  the  ordinary  media,  though  its  growth  on 

house  exert  a  very  prejudicial  effect  upon  tubercular  patients.  Sir  Richard 
Thome  Thorne 1  has  described  the  favourable  house  for  such  persons  as  one 
built  upon  a  soil  which  is  dry  naturally  or  freed  by  artificial  means  from  the 
injurious  influences  of  dampness  and  of  the  oscillations  of  the  underlying  sub- 
soil. The  house  itself  should  be  so  constructed  as  to  be  protected  against 
dampness  of  site,  foundations,  and  walls.  Upon  at  least  two  opposite  sides  of 
the  dwelling-house  there  should  be  enough  open  space  to  secure  ample  move- 
ment of  air  about  it,  and  free  exposure  to  sunlight.  Lastly,  it  should  be  pos- 
sible to  have  free  movement  of  air  by  day  and  night  through  all  habitable 
rooms  of  the  house.  It  is  clear  that  many  inhabited  houses  could  not  stand  to 
these  tests  ;  but  effort  should  be  made  to  approach  as  near  to  such  a  standard 
as  possible. 

17.  Sunlight  and  fresh  air  are  the  greatest  enemies  to  infection. 

18.  Disinfection  is  necessary  after  death  from  phthisis,  and  should  be  as 
complete  as  after  any  other  infective  disease.     Compulsory  notification  of  fatal 
cases  and  compulsory  disinfection  have  been  officially  ordered  by  the  Prussian 
Government.     In  this  country  also  absolute  disinfection  should  always  be  in- 
sisted upon  after  phthisis.     Walls,  floors,    carpets,   curtains,  etc.,  should  be 
strictly  sterilised.     Professor  Delepine  recommends  spraying  with  i-ioo  solu- 
tion of  chloride  of  lime. 

1 Practitioner,  vol.  xlvi. 


BACTERIA    AND  DISEASE  293 

potato  is  not  visible ;  on  the  white  of  egg  it  flourishes  ex- 
tremely well. 

It  retains  its  vitality  in  cultures  and  sometimes  in  the 
throat  for  months.  Three  or  four  weeks  is  the  average 
length  of  time  for  its  existence  in  the  membrane,  but,  owing 
to  the  difficulty  of  killing  it  in  situ,  it  may  live  on  for  as  long 
as  a  year.  All  the  conditions  in  the  throat — mucous  mem- 
brane, blood-heat,  moisture,  air — are  extremely  favourable 
to  the  bacillus;  but  it  is  very  materially  modified  in  viru- 
lence. It  is  secured  for  diagnostic  purposes  by  one  of  two 
methods:  (a)  Either  a  piece  of  the  membrane  is  detached, 
and  after  washing  carefully  examined  by  culture  as  well  as 
the  microscope;  or  (f)  a  "  swab  "  is  made  from  the  infected 
throat  and  cultured  on  serum,  and  incubated  at  37°  C.  for 
eighteen  hours  and  then  microscopically  examined.  Both 
methods — and  there  is  no  further  choice — present  some 
difficulties  owing  to  the  large  number  of  bacteria  found  in 
the  throat.  Hence  a  negative  result  must  be  accepted  with 
reserve. 

We  have  already  referred  at  some  length  to  the  question 
of  toxins  in  diphtheria,  and  need  not  dwell  further  upon 
that  matter.  Still  a  word  or  two  may  be  said  here  sum- 
marising the  general  action  of  the  bacillus.  Locally  it  pro- 
duces inflammatory  change  with  fibrinous  exudation  and 
some  cellular  necrosis.  In  the  membrane  a  ferment  is 
probably  produced  which,  unlike  the  localised  bacilli,  passes 
throughout  the  body  and  by  digestion  of  the  proteids  pro- 
duces albumoses  and  an  organic  acid  which  have  the  toxic 
influence.  The  toxins  act  on  the  blood-vessels,  and  nerves, 
and  muscle  fibres  of  the  heart,  and  many  of  the  more  highly 
specialised  cells  of  the  body.  Thus  we  get  degenerative 
changes  in  the  kidney,  in  cells  of  the  central  nervous  system, 
in  the  peripheral  nerves  (post-diphtheritic  paralysis),  and 
elsewhere,  these  pathological  conditions  setting  up,  in  ad- 
dition to  the  membrane,  the  signs  of  the  disease.  The  bacil- 


294  BACTERIA 

lus  is  pathogenic  for  the  horse,  ox,  rabbit,  guinea-pig,  cat, 
and  some  birds.  Cases  are  on  record  of  supposed  infection 
of  children  by  cats  suffering  from  the  disease.  The  horse, 
it  will  be  remembered,  yields  the  antitoxin  which  has  saved 
so  many  lives  (Metropolitan  Asylums  Board  Report,  1896). 

The  influence  of  drainage,  milk,  and  schools  must  not  be 
forgotten  by  sanitary  authorities  any  more  than  the  essen- 
tial importance  of  adequate  isolation  hospital  accommoda- 
tion. Mr.  Shattock's  experiments  on  the  effect  of  sewer  air 
upon  attenuated  Klebs-Loffler  bacilli  have  been  mentioned 
(see  p.  105).  Nevertheless  there  can  be  no  doubt  that 
emanations  from  defective  drains  have  a  materially  predis- 
posing effect,  not,  it  is  true,  upon  the  bacilli,  but  upon  the 
tissues.  Sore  throats  thus  acquired  are  par  excellence  the 
site  for  the  development  of  diphtheria. 

The  influence  of  school  attendance  has  claimed  the  recent 
attention  of  the  Medical  Officer  of  the  London  School  Board 
and  the  Medical  Officer  of  the  administrative  County  of 
London.  In  London  since  1881  there  has  been  a  marked 
increase  of  diphtheria,  which  has  occurred,  though  in  a  much 
less  degree,  throughout  England  and  Wales. 

The  Registrar-General  has  only  classified  diphtheria  as  a 
separate  disease  since  1855,  when  the  death-rate  per  1,000,. 
ooo  in  England  and  Wales  was  stated  as  20.  The  following 
are  the  figures  for  four  decades  up  to  1895  : 

AVERAGE  DEATH-RATE  PER  MILLION  OF  THE  POPULATION 
FROM  DIPHTHERIA  IN  ENGLAND  AND  WALES  AND  IN 
LONDON  (IN  DECADES  1856-95) 

England  and 

Wales.  London. 

1856-65 246.9  225.4 

1865-75 124.8  123.5 

I875~^5 129.0  176.7 

1885-95 210.6  421.4 


BACTERIA   AND  DISEASE  29$ 

From  these  figures  the  extraordinary  increase  during  the 
last  few  years  is  clearly  demonstrated. 

Sir  Richard  Thorne  Thorne,  in  1891,  drew  attention  to 
the  influence  of  damp  soils  and  schools  upon  diphtheria. 
In  1894  Mr.  Shirley  Murphy,  Medical  Officer  to  the  London 
County  Council,  reported  that  there  had  been  an  increase  in 
diphtheria  mortality  in  London  at  school  ages  (three  to  ten) 
as  compared  with  other  ages  since  the  Elementary  Education 
Act  became  operative  in  1871 ;  that  the  increased  mortality 
from  diphtheria  in  populous  districts,  as  compared  with  rural 
districts,  since  1871,  might  be  due  to  the  greater  effect  of 
the  Education  Act  in  the  former;  and  that  there  was  a 
diminution  of  diphtheria  in  London  during  the  summer 
holidays  at  the  schools  in  1893,  but  that  1892  did  not  show 
any  marked  changes  for  August. 

In  1896  Professor  W.  R.  Smith,  the  Medical  Officer  to  the 
London  School  Board,  furnished  a  report 1  on  this  same 
subject  of  school  influence,  in  which  he  produces  evidence 
to  show  that  the  recrudescence  of  the  disease  in  1881-90  was 
greatest  in  England  and  Wales  at  the  age  of  two  to  three 
years,  and  in  London  at  the  age  of  one  to  two  years,  in 
both  cases  before  school  age ;  that  age  as  an  absolute  factor 
in  the  incidence  of  the  disease  is  enormously  more  active 
than  any  school  influence,  and  that  personal  contact  is 
another  important  source  of  infection. 

Although  it  is  said  that  "  statistics  can  be  made  to  prove 
anything,"  there  can  be  little  doubt  that  both  of  these 
reports  contain  a  great  deal  of  truth ;  nor  are  these  truths 
incompatible  with  each  other.  They  both  emphasise  age  as 
a  great  factor  in  the  incidence  of  the  disease,  and  whatever 
affects  the  health  of  the  child  population,  like  schools,  must 
play,  directly  or  indirectly,  a  not  unimportant  part  in  the 
transmission  of  the  disease. 

1  Journal  of  State  Medicine,  vol.  iv.  (1896),  p.  169. 


296  BACTERIA 

The  Pseudo-diphtheria  Bacillus.1  LofHer  and  Hoffman 
described  a  bacillus  having  the  same  morphological  charac- 
ters as  the  true  Bacillus  diphtheria,  except  that  it  had  no 
virulence.  Roux  believes  this  is  merely  an  attenuated  diph- 
theria bacillus.  It  is  frequently  found  in  healthy  throats. 
The  chief  differences  between  the  real  and  the  pseudo- 
bacillus  are : 

1.  The  pseudo-bacillus  is  thicker  in  the  middle  than  at 
the  poles,   and  not  so  variable  as  the  Bacillus  dipJitheria. 
Polar  staining  is  absent. 

2.  Its  growth  on  potato  reveals  cream-coloured  colonies 
visible  in  a  couple  of  days;  the  real  bacillus  is  invisible. 

3.  The  pseudo-bacillus  will  not  grow  at  all  anaerobically 
in  hydrogen,  but  the  Bacillus  diphtheria  is  able  to  do  so. 

4.  There  is  the  great  difference  in  virulence. 
Suppuration.     This  term  is  used  to  designate  that  general 

breaking  down  of  cells  which  follows  acute  inflammation. 
An  "  abscess  "  or  "  gathering  "  is  a  collection,  greater  or 
smaller,  of  the  products  of  suppuration.  The  word  pus  is 
generally  used  to  describe  this  matter.  We  may  have  such 
an  advanced  inflammatory  condition  in  any  locality  of  the 
body,  and  it  will  assume  different  characters  according  to 
its  site.  Hence  there  are  connected  with  suppuration,  as 
causal  agents,  a  variety  of  bacteria.  Pus  is  not  matter  con- 
taining a  pure  culture  of  any  specific  species,  but,  on  the 
contrary,  is  generally  filled  with  a  large  number  of  different 
species.  The  most  important  are  as  follows : 

I.  Staphylococcus pyogenes  aureus.  These  are  micrococci 
arranged  in  groups,  which  have  been  likened  to  bunches  of 
grapes.  They  are  the  common  organisms  found  in  pus,  and 
were  with  other  auxiliary  bacteria  first  distinguished  as  such 
by  Professor  Ogston,  of  Aberdeen.  There  are  several  forms 
of  the  same  species,  differing  from  each  other  in  colour. 

1  For  a  fuller  statement  see  Trans.  Jenner  Institute  (First  Series),  pp.  7-32. 


BACTERIA    AND   DISEASE  297 

Thus  we  have  the  5.  pyogenes  aureus  (golden  yellow),  albus 
(white),  citreus  (lemon),  and  others.  They  occur  commonly 
in  nature,  in  air,  soil,  water,  on  the  surface  of  the  skin,  and 
in  all  suppurative  conditions.  The  aureus  is  the  only  one 
credited  with  much  virulence.  It  occurs  in  the  blood  in 
blood-poisoning  (septicaemia,  pyaemia),  and  is  present  in  all 
ulcerative  conditions,  including  ulcerative  disease  of  the 
valves  of  the  heart. 

The  Staphylococcus  cereus  albus  and  5.  cereus  flavus  are 
slightly  modified  forms  of  the  5.  pyogenes  aureus,  and  are 
differentiated  from  it  by  being  non-liquefying.  They  pro- 
duce a  wax-like  growth  on  gelatine. 

Staphylococcus  pyogenes  aureus,  the  type  of  the  family,  is 
grown  in  all  ordinary  media  at  room  temperature,  though 
more  rapidly  at  37°  C.  Liquefaction  sets  in  at  a  compara- 
tively early  date,  and  subsequently  we  have  in  the  gelatine 
test-tube  cultures  a  flocculent  deposit  of  a  bright  yellow 
amorphous  mass,  and  in  gelatine  plates  small  depressions 
of  liquefaction  with  a  yellow  deposit.  It  renders  all  media 
acid,  and  coagulates  milk.  Its  thermal  death-point  in  gela- 
tine is  58°  C.  for  ten  minutes,  but  when  dry  considerably 
higher.  It  is  a  non-motile  and  a  facultative  anaerobe;  but 
the  presence  of  oxygen  is  necessary  for  a  bright  colour.  Its 
virulence  readily  declines. 

2.  Streptococcus  pyogenes.  In  this  species  of  micrococcus 
the  elements  are  arranged  in  chains.  Most  of  the  strepto- 
cocci in  pus,  from  different  sources,  are  one  species,  having 
approximately  the  same  morphological  and  biological  charac- 
ters. Their  different  effects  are  due  to  different  degrees  of 
toxic  virulence;  they  are  always  more  virulent  when  asso- 
ciated with  other  bacteria,  for  example,  the  Proteus  family. 

The  chains  vary  in  length,  consisting  of  more  elements 
when  cultured  in  fluid  media.  They  multiply  by  direct 
division  of  the  individual  elements,  and  in  old  cultures  it 
has  been  observed  that  the  cocci  vary  in  form  and  size. 


298  BACTERIA 

This  latter  fact  gave  support  to  the  theory  that  streptococ- 
cus reproduced  itself  by  arthrospores,  or  "  mother-cells." 

In  culture  upon  the  ordinary  media  streptococcus  is  com- 
paratively slow-growing,  producing  minute  white  colonies 
on  or  about  the  sixth  day.  It  does  not  liquefy  gelatine, 
and  remains  strictly  localised  to  the  track  of  the  inoculating 


TYPES  OF  STREPTOCOCCUS 

needle.  Like  the  staphylococcus,  it  readily  loses  virulence. 
The  thermal  death-point  is,  however,  lower :  54°  C.  for  ten 
minutes.  Marmorek  has  devised  a  method  by  which  the 
virulence  may  be  greatly  increased,  and  he  holds  that  it  de- 
pends upon  the  degree  of  virulence  possessed  by  any  partic- 
ular streptococcus  as  to  what  effects  it  will  produce.  By  the 


BACTERIA   AND  DISEASE  299 

adoption  of  Marmorek's  methods  attempts  have  been  made 
to  prepare  an  antitoxin. 

Streptococcus  pyogenes  has  been  isolated  from  the  mem- 
brane of  diphtheria,  and  from  small-pox,  scarlet  fever,  vac- 
cinia, and  other  diseases.  In  such  cases  it  is  not  the  causal 
agent,  but  merely  associated  with  the  complications  of  these 
diseases.  Suppuration  and  erysipelas  are  the  only  patho- 
logical conditions  in  which  the  causal  agency  of  streptococ- 
cus has  been  sufficiently  established. 

3.  The  Bacillus  pyocyaneus  occurs  in  green  pus,  and  is 
the  cause  of  that  colouration.  Gessard  was  the  first  to  prove 
its  significance,  and  he  describes  two  varieties. 

It  is  a  minute,  actively  motile,  non-sporulating  bacillus, 
which  occasionally  complicates  suppuration  and  produces 


r.:<       *H! 
I?          **        *l# 


MICROCOCCUS  TETRAGONUS 

green  pus.  Oxygen  is  necessary  for  pigmentation,  which  is 
due  to  two  substances :  pyocyanin,  a  greenish-blue  product 
extracted  with  chloroform,  and  pyoxanthose,  a  brown  sub- 
stance derived  from  the  oxidation  of  the  former  pigment. 
Both  these  colours  are  produced  in  cultivation  outside  the 
body.  On  gelatine  the  colour  is  green,  passing  on  to  olive. 
There  is  liquefaction.  On  potato  we  generally  obtain  a 
brown  growth  (compare  Bacillus  coli,  B.  mallei,  and  others). 
The  organism  grows  rapidly  on  all  the  ordinary  media, 
which  it  has  a  tendency  to  colour  throughout. 

It  will  be  remembered  that  when  speaking  of  the  antagon- 
ism of  organisms,  we  referred  to  the  inimical  action  of 
Bacillus  pyocyaneus  upon  anthrax. 

4.  Micrococcus  Tetragonus.    This  species  occurs  in  phthisi- 


300 


BACTERIA 


cal  cavities  and  in  certain  suppurations  in  the  region  of  the 
face.  It  is  a  micrococcus  usually  in  the  form  of  small 
tetrads.  A  capsule  is  always  present  and  sometimes  dis- 
cernible. 

5.  Bacillus  coli  communis  and  many  putrefactive  germs 
commonly  occur  in  suppurative  conditions,  but  they  are  not 
restricted  to  such  disorders  (see  p.  64). 

6.  Micrococcus  gonorrhoea  (Neisser,  1879).     This  organism 
is  more  frequently  spoken  of  as  a  diplococcus.      It  occurs  at 
the  acute  stage   of  the  disease,  but  is  not  readily  differ- 
entiated from  other  similar  diplococci  except  by  technical 


DIPLOCOCCUS  OF  NEISSER 

laboratory  methods.  Each  element  presents  a  straight  or 
concave  surface  to  its  fellow.  A  very  marked  concavity 
indicates  commencing  fission.  The  position  which  these 
diplococci  take  up  in  pus  is  intracellular,  and  arranged 
more  or  less  definitely  around  the  nucleus.  Difficulty  has 
often  been  found  in  cultivating  this  organism  in  artificial 
media  outside  the  body.  Wertheim  and  others  have  sug- 
gested special  formulae  for  the  preparation  of  suitable  media, 
but  it  is  a  very  simple  matter  to  secure  cultures  on  agar 
plates  smeared  with  human  blood  from  a  pricked  finger. 
The  plate  is  incubated  at  37°  C.  At  the  end  of  twenty-four 
hours  small  raised  grey  colonies  appear,  which  at  the  end  of 


BACTERIA   AND  DISEASE  301 

about  four  days  show  adult  growth.  The  margin  is  un- 
even, and  the  centre  more  opaque  than  the  rest  of  the 
colony.  This  diplococcus  is  readily  killed,  and  sub-cultures 
must  be  frequently  made  to  retain  vitality  and  virulence. 
Light,  desiccation,  and  a  temperature  of  55°  C.  all  act  germ- 
icidally.  The  organism  stains  readily  in  Loffler's  blue,  but 
is  decolourised  by  Gram's  method.  It  is  more  or  less 
strictly  parasitic  to  man.  Its  shape,  size,  character  of 
growth,  and  staining  properties  assist  in  differentiating  it 
from  various  similar  diplococci.1 

Anthrax.  This  disease  was  one  of  the  first  in  which  the 
causal  agency  of  bacteria  was  proved.  In  1849  Pollender 
found  an  innumerable  number  of  small  rods  in  the  blood  of 
animals  suffering  from  anthrax.  In  1863  Davaine  described 
these,  and  attributed  the  disease  to  them.  But  it  was  not 
till  1876  that  Koch  finally  settled  the  matter  by  isolating  the 
bacilli  in  pure  culture  and  describing  their  biological  char- 
acters. 

It  is  owing  in  part  to  its  interesting  bacterial  history, 
which  opened  up  so  much  new  ground  in  this  comparatively 
new  science,  that  anthrax  has  assumed  such  an  important 
place  in  pathology.  But  for  other  reasons,  too,  it  claims 
attention.  It  appears  to  have  been  known  in  the  time  of 
Moses,  and  was  perhaps  the  disease  described  by  Homer  in 
the  First  Book  of  the  Iliad.  Rome  was  visited  by  it  in 
740  B.C. 

Anthrax  is  an  acute  disease,  affecting  sheep,  cattle, 
horses,  goats,  deer,  and  man.  Cats,  white  rats,  and  Alger- 
ian sheep  are  immune.  Swine  become  infected  by  feeding 
on  the  offal  of  diseased  cattle  (Crookshank). 

The  post-mortem  signs  are  mainly  three:  The  spleen  is 
greatly  enlarged  and  congested,  is  friable  to  the  touch,  and 
contains  enormous  numbers  of  bacilli ;  the  skin  may  show 
exudations  forming  dark  gelatinous  tumours ;  and  the  blood 

1  See  Trans.  Jenner  Institute  (First  Series),  A.  G.  R.  Foulerton,  pp.  40-81. 


302 


BACTERIA 


remains  fluid  for  some  time  after  death,  is  black,  tar-like, 
contains  bubbles  of  air,  and  shows  other  degenerative 
changes  in  the  red  corpuscles,  whilst  the  small  blood-vessels 
contain  such  vast  quantities  of  bacilli  that  they  may  be 
ruptured  by  them.  Particularly  is  this  true  in  the  peri- 
pheral arteries.  Many  of  the  organs  of  the  body  show 
marked  congestion. 

Clinically  there  is  rise  of  temperature  and  rapid  loss  of 
muscular  power.  The  bacilli  of  anthrax  are  square-ended 
rods  i  ^  broad  and  4-5  /*  long.  In  the  tissues  of  the  body 
they  follow  the  lines  of  the  capillaries,  and  are  irregularly 


BACILLUS  OF  ANTHRAX  AND 
BLOOD  CORPUSCLES 


THREADS  OF  BACILLUS 
ANTHRACIS,  SHOWING  SPORES 


situated.  In  places  they  are  so  densely  packed  as  to  form 
obstructions  to  the  onward  flow  of  blood.  In  cultures  they 
are  in  chains  end  to  end,  having  as  a  rule  equal  interbacil- 
lary  spaces.  In  cultures  long  filaments  and  threads  occur. 
The  exact  shape  of  the  bacillus  depends,  however,  upon 
two  things:  the  staining  and  spore  formation.  Both  these 
factors  may  very  materially  modify  the  normal  shape.  The 
spores  of  anthrax  are  oval  endospores,  produced  only 'in  the 
presence  of  free  oxygen,  and  at  any  temperature  between 
1 8  and  41°  C.  On  account  of  requiring  free  oxygen,  they 
are  formed  only  outside  the  body.  The  homogeneous  pro- 


BACTERIA    AND  DISEASE  303 

toplasm  of  the  bacillus  becomes  granular;  the  granules 
coalesce,  and  we  have  spores.  Each  spore  possesses  a  thick 
capsule,  which  enables  it  to  resist  many  physical  conditions 
which  kill  the  bacillus.  When  the  spore  is  ripe  or  has  ex- 
hausted the  parent  bacillus,  it  may  take  on  a  resting  stage, 
or  under  favourable  circumstances  commence  germination, 
very  much  after  the  manner  of  a  seed.  The  spores  may  in- 
fect a  farm  for  many  months;  indeed,  cases  are  on  record 
which  appear  to  prove  that  the  disease  on  a  farm  in  the 
autumn  may  by  means  of  the  spores  be  carried  on  by  the 
hay  of  the  following  summer  into  a  second  winter.  Thus, 
by  means  of  the  spores,  the  infection  of  anthrax  may  cling 
to  the  land  for  very  long  periods,  even  for  years.  Spores 
of  anthrax  can  withstand  5  percent,  carbolic  acid  or  i-iooo 
corrosive  sublimate  for  more  than  an  hour;  even  boiling 
does  not  kill  them  at  once,  whilst  the  bacilli  without  their 
spores  are  killed  at  54°  C.  in  ten  minutes.  When  the  spores 
are  dry  they  are  much  more  resistant  than  when  moist. 
Hence  the  persistence  of  the  anthrax  bacillus  is  due  to  its 
spores. 

The  bacillus  is  aerobic,  non-motile,  and  liquefying.  Broth 
cultures  become  turbid  in  thirty-six  hours,  with  nebulous 
masses  of  threads  matted  together.  The  pellicle  which 
forms  on  the  surface  affords  an  ideal  place  for  spore  form- 
ation. 

Cultures  in  the  depth  of  gelatine  show  a  most  character- 
istic growth.  From  the  line  of  inoculation  delicate  threads 
and  fibrillae  extend  outwards  horizontally  into  the  medium. 
Liquefaction  commences  at  the  top,  and  eventually  extends 
throughout  the  tube.  On  gelatine  plates  small  colonies 
appear  in  thirty-six  hours,  and  on  the  second  or  third  day 
they  look,  under  a  low  power  of  the  microscope,  like  matted 
hair.  The  colonies  after  a  time  sink  in  the  gelatine,  owing 
to  liquefaction.  On  potato,  agar,  and  blood  serum  anthrax 
grows  well. 


304  BACTERIA 

Channels  of  Infection.  I.  The  Alimentary  Canal.  This 
is  the  usual  mode  of  infection  in  animals  grazing  on  infected 
pasture  land.  A  soil  suitable  for  the  propagation  of  anthrax 
is  one  containing  abundance  of  air  and  proteid  material. 
Feeding  on  bacilli  alone  would  probably  not  produce  the 
disease,  owing  to  the  germicidal  effect  of  the  gastric  juice. 
But  spores  can  readily  pass  uninjured  through  the  stomach 
and  produce  anthrax  in  the  blood.  Infected  water  as  well 
as  fodder  may  convey  the  disease.  Water  becomes  infected 
by  bodies  of  animals  dead  of  anthrax,  or,  as  was  the  case 
once  at  least  in  the  south-west  of  England,  by  a  stream 
passing  through  the  washing-yard  of  an  infected  tannery. 
Manure  on  fields,  litter  in  stalls,  and  infected  earth  may  all 
contribute  to  the  transmission  of  the  disease.  Darwin 
pointed  out  the  services  which  are  performed  in  superficial 
soils  by  earthworms  bringing  up  casts;  Pasteur  was  of 
opinion  that  in  this  way  earthworms  were  responsible  for 
continually  bringing  up  the  spores  of  anthrax  from  buried 
corpses  to  the  surface,  where  they  would  reinfect  cattle. 
Koch  disputed  this,  but  more  recently  Bollinger  has  demon- 
strated the  correctness  of  Pasteur's  views  by  isolating  an- 
thrax contagium  from  five  per  cent,  of  the  worms  sent  him 
from  an  anthrax  pasture.  Bollinger  also  maintains  that 
flies  and  other  insects  may  convey  the  disease  from  dis- 
charges or  carcasses  round  which  they  congregate. 

Alimentary  infection  in  man  is  a  rare  form,  and  it  reveals 
itself  in  a  primary  diseased  state  known  as  mycosis  intestin- 
alis,  an  inflamed  condition  of  the  intestine  and  mesenteric 
lymph  glands. 

2.  Through  the  Skin.  Cutaneous  anthrax  goes  by  the 
name  of  malignant  pustule,  and  is  caused  by  infective  an- 
thrax matter  gaining  entrance  through  abrasions  or  ulcers 
in  the  skin.  This  local  form  is  obviously  most  contracted 
by  those  whose  occupation  leads  them  to  handle  hides  or 
other  anthrax  material  (butchers  and  cleaners  of  hides). 


BACTERIA   AND  DISEASE  305 

Two  or  three  days  after  inoculation  a  red  pimple  appears, 
which  rapidly  passes  through  a  vesicular  stage  until  it  is  a 
pustule.  Concomitantly  we  have  glandular  enlargement, 
general  malaise,  and  a  high  temperature.  Thus  from  a  local 
sore  a  general  infection  may  result.  Unless  this  does  occur, 
the  issue  will  not  be  fatal,  and  the  bacilli  will  never  gain 
entrance  into  the  blood  or  be  anything  but  local. 

3.  Respiratory  Tract.  In  man  this  is  the  commonest  form 
of  all,  and  is  well  known  under  the  term  "  wool-sorters'  dis- 
ease, ' '  or  pulmonary  anthrax.  This  mode  of  infection  occurs 
when  dried  spores  are  inhaled  in  processes  of  skin-cleaning. 
It  frequently  commences  as  a  local  lesion  affecting  the 
mucous  membrane  of  the  trachea  or  bronchi,  but  it  rapidly 
spreads,  affecting  the  neighbouring  glands,  which  become 
greatly  enlarged,  and  extending  to  the  pleura  and  lung 
itself.  Such  cases,  as  a  rule,  rapidly  end  fatally. 

From  what  has  been  said,  it  will  be  clear  that  anthrax 
carcasses  are  better  not  opened  and  exposed  to  free  oxygen. 
An  extended  post-mortem  examination  is  not  necessary. 
Burning  the  entire  carcass  in  a  crematorium  would  be  the 
ideal  treatment.  As  such  is  not  generally  feasible  the  next 
best  thing  is  to  bury  the  carcass  deeply  with  lime  below  and 
above  it,  and  rail  in  the  area  to  prevent  other  animals  graz- 
ing off  it. 

A  very  small  prick  will  extract  enough  blood  to  examine 
for  the  anthrax  bacilli  which  are  driven  by  the  force  of  the 
blood-current  to  the  small  surface  capillaries.  This  occurs, 
of  course,  only  when  the  disease  has  become  quite  general, 
for  in  the  early  stage  the  healthy  blood  limits  the  bacilli 
to  the  internal  organs.  In  such  cases  examination  of  the 
blood  of  the  spleen  is  necessary. 

Anthrax  covers  a  wide  geographical  area  all  over  the 
world,  and  no  country  seems  altogether  exempt.  In  Ger- 
many as  many  as  3700  animals  have  been  lost  in  a  single  year. 
About  900  animals  were  attacked  in  1897  in  Great  Britain. 


306  BACTERIA 

Plague.  This  disease,  like  anthrax  and  leprosy,  has  a 
long  historical  record  behind  it.  As  the  Black  Death  it 
decimated  the  population  of  England  in  the  fourteenth 
century,  and  visited  the  country  again  in  epidemic  form  in 
the  middle  of  the  seventeenth  century,  when  it  was  called 
the  Great  Plague.  Now,  it  is  highly  probable  that  these  two 
scourges  and  the  recent  epidemic  in  the  East  are  all  forms 
of  one  and  the  same  disease.  As  a  matter  of  fact,  it  is 
difficult  to  be  sure  what  was  the  exact  pathology  of  a  num- 
ber of  the  grievous  ailments  which  troubled  our  country  in 
the  Middle  Ages,  but  from  all  accounts  bubonic  plague  and 
true  leprosy  were  amongst  them.  The  former  came  and 


7'" 

**  *-\ 

BACILLUS  OF  PLAGUE 

went   spasmodically,    as   is   its    habit;    the    latter   dragged 
through  the  length  of  several  centuries. 

The  distribution  of  plague  at  the  present  time  is  fort- 
unately a  somewhat  limited  one,  namely,  a  definite  area  in 
Asia  known  as  the  "  Plague  Belt."  From  Mesopotamia, 
as  a  sort  of  focus,  the  disease  spreads  northwards  to  the 
Caspian  Sea,  westwards  to  the  Red  Sea,  southwards  as  far 
as  Central  India,  and  eastwards  as  far  as  the  China  Sea. 
This  constitutes  the  "  belt,"  but  the  disease  may  take  an 
epidemic  form,  and  is  readily,  though  very  slowly,  conveyed 
by  infection  or  contagion.  It  appears  to  be  infectious  by 
means  of  infective  dust,  and  contagious  by  prolonged  and 
intimate  contact  with  the  plague-stricken.  Rats  have  been 
accused  of  conveying  the  disease  from  port  to  port,  and 


BACTERIA   AND  DISEASE  307 

even  infecting  man.  It  is  clear  that  rats  are  not  the  only 
agency  acting  in  this  way.  Nevertheless  it  is  true  that  rats 
contract  the  disease  more  readily  than  any  other  animals, 
and  that  when  suffering  from  it  they  may  spread  the  in- 
fection. How  it  is  thus  spread  it  is  not  known.  Drs. 
Cantlie  and  Yersin  have  pointed  out  that  previously  to  an 
epidemic  of  plague  rats  die  in  enormous  numbers. 

The  bacteriology  of  plague  is  almost  the  latest  addition 
to  the  science.  Kitasato,  of  Tokio,  demonstrated  the  cause 
of  plague  to  be  a  bacillus  during  the  Hong  Kong  epidemic 
in  1894.  This  was  immediately  confirmed  by  Yersin,  and 
further  proved  by  the  isolation  in  artificial  media  of  a  pure 
culture  of  a  bacillus  able  to  cause  the  specific  disease  of 
bubonic  plague. 

The  bacillus  was  first  detected  in  the  blood  of  patients 
suffering  from  the  disease.  It  takes  the  form  of  a  small, 
round-ended,  oval  cell,  with  marked  polar  staining,  and 
hence  having  an  appearance  not  unlike  a  diplococcus.  In 
the  middle  there  is  a  clear  interspace,  and  the  whole  is  sur- 
rounded with  a  thick  capsule,  stained  only  with  difficulty. 
The  organisms  are  often  linked  together  in  pairs  or  even 
chains,  and  exhibit  involution  forms.  In  culture  the  bacil- 
lus is  even  more  coccal  in  form  than  in  the  body. 

The  plague  bacillus  grows  readily  on  the  ordinary  media 
at  blood-heat,  producing  circular  cream-coloured  colonies 
with  a  wavy  outline,  which  eventually  coalesce  to  form  a 
greyish  film.  The  following  negative  characters  help  to 
distinguish  it :  No  growth  occurs  on  potato,  milk  is  not 
coagulated,  and  gelatine  is  not  liquefied ;  Gram's  method 
does  not  stain  the  bacillus,  and  there  are  no  spores;  the 
bacillus  is  readily  killed  by  heat  and  by  desiccation  over 
sulphuric  acid  at  30°  C.  Both  in  cultures  and  outside  the 
body  the  bacillus  loses  virulence.  To  this  may  be  attributed 
possibly  the  variety  of  forms  of  the  plague  bacillus  which 
differ  in  virulence.  On  gaining  entrance  to  the  human  body 


308  BACTERIA 

the  bacillus  affects  in  particular  two  organs,  the  spleen  and 
the  lymph  glands.  The  latter  become  inflamed  in  groups, 
commencing  frequently  with  those  in  the  armpit  (axillary) 
or  groin  (inguinal).  The  spleen  suffers  from  inflammatory 
swelling,  which  may  affect  other  organs  also.  In  both 
places  the  bacilli  occur  in  enormous  numbers.  Kitasato 
considers  that  the  bacillus  may  enter  the  body  by  the  three 
channels  adopted  by  anthrax,  namely,  the  skin,  alimentary 
canal,  and  respiratory  tract. 

Haffkine  has  prepared  a  vaccine  to  be  used  as  a  prophy- 
lactic. He  grows  a  pure  culture  of  Kitasato's  bacillus  in 
broth  upon  the  surface  of  which  some  globules  of  fat 
("  ghee  ")  have  been  placed.  The  bacillus  grows  upon  this 
fat  in  copious  stalactite  form.  From  time  to  time  this 
growth  is  shaken  down,  until  after  five  or  six  weeks  the 
shaken  broth  appears  milky.  The  contained  bacilli  are 
killed  by  heating  the  fluid  to  70°  C.  for  one  hour.  The  re- 
sultant is  the  vaccine,  of  which  the  dose  is  3  cc.  Haffkine 
believes  that  inoculated  persons  in  India  have  suffered 
twenty  times  less  than  non-inoculated  living  under  the  same 
conditions. 

Plague  is  essentially  a  "  filth  disease,"  and  it  is  frequently 
preceded  by  famine.  Temperature  and  overcrowding  exert 
an  influence  upon  it.  The  areas  affected  by  the  disease  in 
the  Middle  Ages,  in  the  seventeenth  century,  and  in  1894- 
96  are  alike  in  being  characterised  by  filth  and  overcrowd- 
ing. There  is  little  fear,  speaking  generally,  of  the  plague 
ever  flourishing  under  Western  civilisation,  where  the  con- 
ditions are  such  that  even  when  it  appears  there  is  little  to 
encourage  or  favour  its  development. 

Leprosy.  This  ancient  disease  is  said  to  have  existed  in 
Egypt  3500  B.C.,  and  was  comparatively  common  in  India, 
China,  and  even  in  parts  of  Europe  500  B.C.  We  know  it 
has  existed  in  many  parts  of  the  world  in  the  past,  in  which 
regions  it  is  now  extinct.  Some  of  the  earliest  notices  we 


BACTERIA   AND  DISEASE  309 

have  of  it  in  this  country  come  from  Ireland,  and  date  back 
to  the  fifth  and  sixth  centuries.  Even  at  that  period  of 
time  also  various  classical  descriptions  of  the  disease  had 
been  written  and  various  decrees  made  by  the  Church  coun- 
cils to  protect  lepers  and  prevent  the  spread  of  the  disease, 
which  was  often  looked  upon  as  a  divine  visitation.  In  the 
tenth  century  leprosy  was  prevalent  in  England ;  it  reached 
its  zenith  in  the  thirteenth  century,  or  possibly  a  little 
earlier,  and  declined  from  that  date  to  its  extinction  in  the 
sixteenth.  But  even  two  hundred  years  later  leprosy  was 
endemic  in  the  Shetlands,  and  it  is  recorded  that  in  1742 
there  was  held  a  public  thanksgiving  in  Shetland  on  account 
of  the  disappearance  of  leprosy. 

At  one  time  or  another  there  were  as  many  as  two  hun- 
dred institutions  in  the  British  Isles  for  the  more  or  less 
exclusive  use  of  lepers.  Many  of  these  establishments  were 
of  an  ecclesiastical  or  municipal  character,  and  probably  the 
exact  diagnosis  of  diseases  was  a  somewhat  lax  matter. 
Bury  St.  Edmunds,  Bristol,  Canterbury,  London,  Lynn, 
Norwich,  Thetford,  and  York  were  centres  for  lepers.  Bur- 
ton Lazars  and  Sherburn,  in  Durham,  were  two  of  the 
more  famous  leper  institutions. 

At  the  present  time  the  distribution  of  the  disease  is 
mostly  Asiatic.  Norway  contains  about  1200  lepers,  Spain 
approximately  the  same  number.  Scattered  through  Europe 
are  perhaps  another  600  or  700,  in  India  100,000,  and  a  large 
number  in  Japan.  The  Cape  possesses  a  famous  leper  hos- 
pital on  Robben  Island,  with  a  number  of  patients.  The 
disease  is  also  endemic  in  the  Sandwich  Islands. 

Descriptions  of  the  pathological  varieties  of  leprosy  have 
been  very  diverse.  The  classification  now  generally  adopted 
includes  three  forms:  the  tuberculated,  the  anesthetic  (or 
maculo-anaesthetic),  and  the  mixed.  Lepra  tuberculosa  is 
that  form  of  the  disease  affecting  chiefly  the  skin,  and 
resulting  in  nodular  tuberculated  growth  or  a  diffuse  infil- 


310  BACTERIA 

tration.  It  causes  great  disfigurement.  The  anaesthetic 
form  causes  a  destruction  of  the  nerve  fibres,  and  so  pro- 
duces anaesthesia,  paralysis,  and  what  are  called  "  trophic  " 
changes.  Not  infrequently  patches  occur  on  the  skin,  which 
appear  like  parchment,  owing  to  this  trophic  change.  Bullae 
may  arise.  When  the  tissue  change  is  radical  or  far  ad- 
vanced, considerable  distortion  may  result.  The  mixed 
variety  of  leprosy,  as  its  name  implies,  is  a  mixture  of  the 
two  other  forms. 

The  Bacillus  leprce  was  discovered  by  Hansen  in  1874. 
He  found  it  in  the  lepra  cells  in  the  skin,  lymph  glands, 
liver,  spleen,  and  thickened  parts  of  the  nerves.  It  is  com- 
mon in  the  discharges  from  the  wounds  of  lepers.  It  is 
conveyed  in  the  body  by  the  lymph  stream,  and  has  rarely 
been  isolated  from  the  blood  (Kobner). 

The  bacillus  is  present  in  enormous  numbers  in  the  ski/i 
and  tissues,  and  has  a  form  very  similar  indeed  to  Bacillus 
tuberculosis.  It  is  a  straight  rod,  and  showing  with  some 
staining  methods  marked  beading,  but  with  others  no  bead- 
ing at  all.  It  measures  4  //.  long  and  i  /u,  broad.  Young 
leprosy  bacilli  are  said  to  be  motile,  but  old  ones  are  not. 
Neisser  has  maintained  that  the  bacillus  possesses  a  capsule 
and  spores.  The  latter  have  not  been  seen,  but  Neisser 
holds  that  this  is  the  form  in  which  the  bacillus  gains  en- 
trance to  the  body.  There  is  a  characteristic  which  fort- 
unately  aids  us  in  the  diagnosis  of  this  disease  in  the  tissues, 
and  that  is  the  arrangement  of  the  bacilli,  which  are  rarely 
scattered  or  isolated,  but  gathered  together  in  clumps  and 
colonies.  Bordoni-Uffreduzzi  and  Campania  claim  to  have 
isolated  the  bacillus  and  grown  it  on  artificial  media,  the 
former  aerobically  on  peptone-glycerine-blood-serum,  at 
37°  C.,  the  latter  anaerobically.  But  no  other  worker  has 
been  able  to  do  this.  Hence  we  are  not  able  to  study  the 
bacteriology  of  leprosy  at  all  completely,  nor  have  inocula- 
tion experiments  proved  successful.  Nevertheless  there  is 


IS?-*' 

ffpfl 
•*%•  . 


BACILLUS  OF  PLAGUE  (B.  PESTIS 
BUBONICCE) 

(From  liver  of  rat) 

X  TOCO 
Bv  permission  of  the  Scientific  Fress^  Limited 


BACILLUS  OF  LEPROSY  (HANSEN) 

(From  the  tissues  of  a  leper) 
X  1000 


STREPTOTHRIX  ACTINOMYCES 
(RAY  FUNGUS) 

X  700 


BACILLUS  MALLEI  (GLANDERS) 


X  looo 


BACTERIA    AND  DISEASE  311 

little  doubt  that  leprosy  is  a  bacterial  disease  produced  by 
the  bacillus  of  Hansen.  Bordoni-Uffreduzzi  maintains  that 
the  parasitic  existence  of  the  Bacillus  leprce  may  alternate 
with  a  saprophytic  stage.  This  may  be  of  importance  in 
the  spread  of  the  disease.  There  is  evidence  in  support  of 
the  non-communicability  of  the  disease  by  heredity  or  con- 
tagion. Segregation  does  not  appear  always  to  result  in  a 
decline  of  the  disease,  as  we  should  expect  if  it  were  purely 
contagious.  Ehlers,  of  Copenhagen,  has,  however,  as  re- 
cently as  1897,  reaffirmed  his  belief  in  the  contagiousness  of 
leprosy;  Virchow,  on  the  other  hand,  has  declared  that  it  is 
not  highly  contagious.  There  is  evidence  to  show  that  per- 
sons far  advanced  in  the  disease  may  live  in  a  healthy  com- 
munity and  yet  not  infect  their  immediate  neighbours. 
Indeed,  the  transmission  of  the  disease  is  still  an  unsolved 
problem.  Mr.  Hutchinson  suggests  diet,  particularly  un- 
cooked or  putrid  fish,  as  a  likely  channel;  on  the  other 
hand,  leprosy  appears  in  districts  where  no  fish  is  eaten. 
Deficiency  of  salt,  telluric  and  climatic  conditions,  racial 
tendencies,  social  status,  poverty,  insanitation,  drinking 
water,  even  vaccination,  have  all  secured  support  from 
various  seekers  after  the  true  channel  by  which  the  bacillus 
gains  entrance  to  the  human  body.  The  real  mode  of 
transmission  is,  however,  still  unknown.  The  decline  and 
final  extinction  of  leprosy  in  the  British  Islands  was  prob- 
ably due  in  part  to  the  natural  tendency  of  the  disease, 
under  favourable  hygienic  circumstances,  to  die  out,  and  in 
part  to  a  general  and  extensive  social  improvement  in  the 
life  of  the  people,  to  a  complete  change  in  the  poor  and  in- 
sufficient diet,  and  to  agricultural  advancement,  improved 
sanitation,  and  land  drainage. 

At  the  Leprosy  Congress  held  in  Berlin  in  1897,  Hansen 
again  emphasised  his  belief  that  segregation  was  the  cause 
of  the  decline  of  leprosy  wherever  it  had  occurred.  But 
there  appears  to  be  some  evidence  to  show  that  leprosy  has 


312  BACTERIA 

declined  where  there  has  been  no  segregation  whatever,  and 
therefore,  however  favourable  to  decline  such  isolation  may 
be,  it  would  seem  not  to  be  actually  necessary  to  the  de- 
cline. At  the  same  Congress  Besnier  declared  in  favour  of 
the  infective  virus  being  widely  propagated  by  means  of  the 
nasal  secretion.  Sticker  states  that  the  nasal  secretion  con- 
tains millions  of  lepra  bacilli,  especially  in  the  acute  stage 
of  the  disease,  and  Besnier  and  Sticker  have  pointed  out  how 
frequently  and  severely  the  septum  nasi  and  skin  over  the 
nose  are  affected  in  leprosy.  Several  leprologists  in  India 
have  recorded  similar  observations.  These  facts  appear  to 
support  Besnier's  contention  that  the  disease  is  spread  by 
nasal  secretion. 

We  may  fitly  add  here  the  conclusions  arrived  at  by  the 
English  Leprosy  Commission  1  in  India: 

i.  Leprosy  is  a  disease  sui  generis ;  it  is  not  a  form  of 
syphilis  or  tuberculosis,  but  has  striking  etiological  analogies  with 
the  latter. 

"  2.  Leprosy  is  not  diffused  by  hereditary  transmission,  and, 
for  this  reason  and  the  established  amount  of  sterility  among 
lepers,  the  disease  has  a  natural  tendency  to  die  out. 

"  3.  Though  in  a  scientific  classification  of  diseases  leprosy 
must  be  regarded  as  contagious,  and  also  inoculable,  yet  the 
extent  to  which  it  is  propagated  by  these  means  is  exceedingly 
small. 

"  4.  Leprosy  is  not  directly  originated  by  the  use  of  any  par- 
ticular article  of  food,  nor  by  any  climatic  or  telluric  conditions, 
nor  by  insanitary  surroundings,  neither  does  it  peculiarly  affect 
any  race  or  caste. 

"5.  Leprosy  is  indirectly  influenced  by  insanitary  surround- 
ings, such  as  poverty,  bad  food,  or  deficient  drainage  or  vent- 
ilation, for  these  by  causing  a  predisposition  increase  the 
susceptibility  of  the  individual  to  the  disease. 

'Dated  1890-91.  The  Commissioners  were  the  late  Beaven  Rake,  M.D., 
G.  A.  Buckmaster,  M.D.,  the  late  Professor  Kanthack,  of  Cambridge,  the  late 
Surgeon-Major  Arthur  Barclay,  and  Surgeon-Major  S.  J.  Thomson. 


BACTERIA    AND  DISEASE  313 

"  6.  Leprosy,  in  the  great  majority  of  cases,  originates  de  novo, 
that  is,  from  a  sequence  or  concurrence  of  causes  and  conditions 
dealt  with  in  the  Report,  and  which  are  related  to  each  other  in 
ways  at  present  imperfectly  known." 

The  practical  suggestions  of  the  Commission  for  prevent- 
ive treatment  included  voluntary  isolation,  prohibition  of 
the  sale  of  articles  of  food  by  lepers,  leper  farms,  orphan- 


DIPLOCOCCUS  OF  PNEUMONIA 

ages,  and  "  improved  sanitation  and  good  dietetic  condi- 
tions "  generally.  Serum-therapy  has  been  attempted  on 
behalf  of  the  French  Academy  of  Medicine,  but  without 
success.  Many  forms  of  treatment  ameliorate  the  miserable 
condition  of  the  leper,  but  up  to  the  present  no  curative 
agent  has  been  found. 

Pneumonia.  Some  of  the  difficulty  which  has  surrounded 
the  bacteriology  of  inflammation  of  the  lungs  is  due  to  the 
confusion  arising  from  supposing  that  attacks  of  the  disease 
differed  only  in  degree.  Pneumonia,  however,  has  various 
forms,  arising  now  from  one  cause,  now  from  another.  The 
specific  or  croupous  pneumonia  is  associated  with  two  organ- 
isms: Fraenkel's  diplococcus  and  Friedlander's  pneumo- 
bacillus.  Several  other  bacteria  have  from  time  to  time 
been  held  responsible  for  pneumonia,  a  streptococcus  re- 


3 14  BACTERIA 

ceiving,  at  one  time,  some  support.  But  whilst  opinion  is 
divided  on  the  role  of  various  extraneous  and  concomitant 
bacteria  in  lung  disease,  importance  is  attached  to  Fraenkel's 
and  Friedlander's  organisms. 

The  diplococcus  of  Fraenkel  is  a  small,  oval  diplococcus 
found  in  the  "  rusty  "  sputum  of  croupous  pneumonia.  It 
is  non-motile,  non-liquefying,  and  aerobic.  When  examined 
from  cultures  the  diplococci  are  frequently  seen  in  chains, 
not  unlike  a  streptococcus,  and  there  is  some  reason  to  sup- 
pose that  this  form  gave  rise  to  the  belief  that  it  was  another 
species ;  when  examined  from  the  tissues  it  possesses  a  cap- 
sule, but  in  culture  this  is  lost.  It  is  difficult  to  cultivate, 
but  grows  on  glycerine  agar  and  blood  serum  at  blood-heat. 
On  ordinary  gelatine  at  room  temperature  it  does  not  grow, 
or,  if  so,  very  slightly.  The  ideal  fluid  is  a  slightly  alkaline 
liquid  medium,  and  in  twenty-four  hours  a  powdery  growth 
will  occur  in  such  broth.  On  potato  there  is  apparently  no 
growth.  It  rapidly  loses  its  virulence  on  solid  media,  and 
is  said  to  be  non-virulent  after  three  or  four  sub-culturings. 
A  temperature  of  54-58°  C.  for  a  few  minutes  kills  the  bac- 
teria, but  not  the  toxin.  This,  however,  is  removed  by 
filtration,  and  is  therefore  probably  intracellular.  It  is 
attenuated  by  heating  to  70°  C. 

Fraenkel's  diplococcus  occurs,  then,  in  the  acute  stage  of 
pneumonia,  in  company  with  streptococci  and  staphylococci. 
It  also  occurs  in  the  blood  in  certain  suppurative  conditions, 
in  pleurisy  and  inflammation  of  the  pericardium,  and  some- 
times in  diphtheria,  and  therefore  it  is  not  peculiar  to  pneu- 
monia. 

There  is  one  other  point  to  which  attention  should  be 
drawn.  Fraenkel's  organism  is  said  to  be  frequently  pre- 
sent in  the  saliva  of  healthy  persons.  Pneumonia  depresses 
the  resistant  vitality  of  the  tissues,  and  thus  affords  to  the 
diplococcus  present  in  the  saliva  an  excellent  nidus  for  its 
growth. 


BACTERIA   AND  DISEASE  315 

Friedlander'  s  Pneumo -bacillus  is  a  capsulated  oval  coccus, 
assuming  the  form  of  a  small  bacillus.  It  is  inconstant  in 
pneumonia,  unequally  distributed,  and  scarce ;  it  is  aerobic, 
and  facultatively  anaerobic;  it  occasionally  occurs  in  long 
forms  and  filaments;  it  is  non-motile,  non-liquefying,  and 
has  no  spores;  it  does  not  stain  by  Gram's  method,  which 
stain  is  therefore  used  for  differential  diagnosis ;  it  will  grow 
fairly  well  in  ordinary  gelatine  at  20°  C.  ;  and  it  is  a  denitri- 


BACILLUS  OF  INFLUENZA 

fying  organism,  and  also  an  actively  fermentative  one,  even 
fermenting  glycerine.  It  is  not  unlike  Bacillus  coli  corn- 
munis,  and  to  distinguish  it  from  that  organism  we  may 
remember  that  the  B.  coli  is  motile,  never  has  a  capsule, 
produces  indol,  and  does  not  ferment  glycerine. 

Influenza.  In  1892,  during  the  pandemic  of  influenza, 
Pfeiffer  discovered  a  bacillus  in  the  bronchial  mucus  of 
patients  suffering  from  the  disease.  It  is  one  of  the  small- 
est bacilli  known,  and  frequently  occurs  in  chains  not  unlike 
a  streptococcus.  Carron  obtained  the  same  organism  from 
the  blood.  In  the  bronchial  expectoration  it  can  retain  its 
virulence  for  as  long  as  a  fortnight,  but  it  is  quickly  de- 
stroyed by  drying.  The  bacillus  is  aerobic,  non-motile,  and 
up  to  the  present  spores  have  not  been  found.  It  grows 
somewhat  feebly  in  artificial  media,  and  readily  dies  out. 
Blood  serum,  glycerine  agar,  broth,  and  gelatine  have  all 
been  used  at  blood-heat.  It  does  not  grow  at  room  tern- 


3l6  BACTERIA 

perature.  Pfeiffer's  bacillus  appears  most  abundantly  at 
the  height  of  the  disease,  and  disappears  with  convalescence. 
It  is  said  not  to  appear  in  any  other  disease. 

Yellow  Fever.  Sternberg  and  Havelburg  have  both 
isolated  bacilli  from  cases  of  yellow  fever;  but  the  organism 
discovered  by  Sanarelli,  the  Bacillus  icter aides,  is  now  ac- 
cepted as  the  causal  agent  of  the  disease.  It  is  a  small, 
short  rod,  with  round  ends,  and  generally  united  in  pairs ; 
it  has  various  pleomorphic  forms;  it  grows  well  on  all  the 
ordinary  media;  it  is  killed  in  sea- water  at  60°  C.,  and  also 
by  direct  sunlight  in  a  few  hours. 

Diarrhoea  of  Infants.  From  time  to  time  different 
organisms  have  been  isolated  in  this  diseased  condition. 
Bacillus  coli  and  B.  enteriditis  sporogenes  (Klein)  have  been 
held  responsible  for  it.  W.  D.  Booker,  of  Johns  Hopkins 
University,  sums  up  an  extended  research  into  the  question 
as  follows : 

"  No  single  micro-organism  is  found  to  be  the  specific  exciter 
of  the  summer  diarrhoea  of  infants,  but  the  affection  is  generally 
to  be  attributed  to  the  result  of  the  activity  of  a  number  of 
varieties  of  bacteria,  some  of  which  belong  to  well-known  species, 
and  are  of  ordinary  occurrence  and  wide  distribution,  the  most 
important  being  the  streptococcus  and  Proteus  vulgaris. 

'  The  first  step  in  the  pathological  process  is  probably  an  injury 
to  the  epithelium  from  abnormal  or  excessive  fermentation,  from 
toxic  products  of  bacteria,  or  from  other  factors. 

"  Bacteria  exert  a  direct  injury  upon  the  tissues  in  some  in- 
stances, whereas  in  others  the  damage  is  brought  about  indirectly 
through  the  production  of  soluble  poisons." 

Actinomycosis.  This  disease  affects  both  animals  and 
man.  As  Professor  Crookshank  points  out,  it  has  long  been 
known  in  this  country,1  but  its  various  manifestations  have 

1  Bacteriology  and  Infective  Diseases  (1896),  p.  144.  Professor  Crookshank's 
Reports  to  the  Agricultural  Department  of  the  Privy  Council  constitute  the 
most  complete  account  of  this  disease  hitherto  published. 


BACTERIA   AND  DISEASE  317 

been  mistaken  for  other  diseases  or  have  received  popular 
names. 

Here  we  can  only  mention  the  most  outstanding  facts 
concerning  the  disease.  It  is  caused  by  the  "  ray  fungus," 
or  Streptothrix  actinomyces,  which,  growing  on  certain 
cereals,  often  gains  entrance  to  the  tissues  of  man  and  beast 
by  lacerations  of  the  mucous  membrane  of  the  mouth,  by 
wounds,  or  by  decayed  teeth.  Barley  has  been  the  cereal 
in  question  in  some  cases.  The  result  of  the  introduction 
of  the  parasite  is  what  is  termed  an  "  infective  granuloma. " 
This  is,  generally  speaking,  of  the  nature  of  an  inflammatory 
tumour  composed  of  round  cells,  epithelioid  cells,  giant 
cells,  and  fibrous  tissue,  forming  nodules  of  varying  sizes. 
In  some  cases  they  develop  to  large  tumours,  in  others  they 
soon  break  down.  Actinomycosis  in  some  ways  closely 
resembles  tuberculosis  in  its  tissue  characters. 

In  the  discharge  or  pus  from  human  cases  of  the  disease 
small  sulphur-yellow  bodies  may  be  detected,  and  these  are 
tufts  of  "  clubs  "  which  are  the  broken-down  rays  of  the 
parasite;  for  in  the  tissues  which  are  affected  the  parasite 
arranges  itself  in  a  radiate  manner,  growing  and  extending 
at  its  outer  margin  and  degenerating  behind.  In  cattle  the 
centre  of  the  old  ray  becomes  caseated,  like  cheese,  or  even 
calcified,  like  a  stone.  In  the  human  disease  abundant 
"  threads  "  are  formed  as  a  tangled  mass  in  the  middle  of 
the  colony.  As  clubs  characterise  the  bovine  actinomy- 
cosis,  so  threads  are  a  feature  of  the  human  form  of  the 
disease.  But  in  both  there  is  a  third  element,  namely, 
small  round  cells,  called  by  some  spores,  by  others  simply 
cocci.  Authorities  are  not  yet  agreed  as  to  the  precise 
significance  and  role  of  these  round  cells.  The  life-history 
of  the  micro-organism  may  be  summed  up  thus : 

"  The  spores  sprout  into  excessively  fine,  straight  or  sinuous, 
and  sometimes  distinctly  spirilliform  threads,  which  branch  irreg- 


318  BACTERIA 

ularly  and  sometimes  dichotomously.  The  extremities  of  the 
branches  develop  the  club-shaped  bodies.  The  clubs  are  closely 
packed  together,  so  that  a  more  or  less  globular  body  is  formed, 
with  a  central  core  composed  of  a  dense  mass  of  threads  " 
(Crookshank). 

Possibly  these  clubs  represent  organs  of  fructification,  and 
produce  the  spores.  These  latter  are,  it  is  believed,  set  free 
in  the  vicinity  of  the  ray,  and  create  fresh  centres  of  disease. 

In  man  the  disease  manifests  itself  in  various  parts  ac- 
cording to  the  locality  of  entrance.  When  occurring  in  the 
mouth  it  attacks  the  lower  jaw  most  frequently.  In  one 
recorded  case  the  disease  was  localised  to  the  bronchi,  and 
did  not  even  extend  into  the  lungs.  It  was  probably  con- 
tracted by  inhalation  of  the  parasite.  The  disease  may 
spread  to  distant  parts  by  means  of  the  blood  stream, 
and  frequently  the  abscesses  are  apt  to  burrow  in  various 
directions. 

In  the  ox  the  disease  remains  much  more  localised,  and 
frequently  occurs  in  the  lower  jaw,  palate,  or  tongue.  In 
the  last  site  it  is  known  as  "  wooden  tongue,"  owing  to 
the  hardness  resulting.  The  skin  and  subcutaneous  tissues 
are  also  a  favourite  seat  of  the  disease,  producing  the  so- 
called  wens  or  clyers  so  commonly  seen  in  the  fen  country 
(Crookshank).  Actinomycosis  in  cattle  is  specially  preval- 
ent in  river  valleys,  marshes,  and  on  land  reclaimed  from 
the  sea.  The  disease  occurs  at  all  seasons,  but  perhaps 
more  commonly  in  autumn  and  winter.  It  is  more  fre- 
quently met  with  in  young  animals.  The  disease  is  prob- 
ably not  hereditary  nor  readily  communicated  from  animal 
to  animal. 

Actinomyces  may  be  cultivated,  like  other  parasitic  dis- 
eases, outside  the  body.  Gelatine,  blood  serum,  agar, 
glycerine  agar,  and  potato  have  been  used  for  this  purpose. 
After  a  few  days  on  glycerine  agar  at  the  temperature  of 
the  blood  little  white  shining  colonies  appear,  which  in- 


BACTERIA   AND  DISEASE  319 

crease  and  coalesce.  In  about  ten  days'  time  the  culture 
often  turns  a  bright  yellow,  though  it  may  remain  white  or 
even  take  on  a  brown  or  olive  tint.  The  entire  mass  of 
growth  is  raised  dry  and  crinkled,  and  composed  almost 
exclusively  of  threads.  In  its  early  stage  small  bacillary 
forms  occur,  and  in  its  later  stage  coccal  forms.  True  clubs 
never  occur  in  pure  cultures,  although  the  threads  may 
occasionally  show  bulbous  endings. 

Glanders  in  the  horse  and  ass,  and  sometimes  by  com- 
munication in  man  also,  is  caused  by  a  short,  non-motile, 
aerobic  bacillus,  named,  after  the  old  Roman  nomenclature 
(malleus),  Bacillus  mallei.  It  was  discovered  in  1882  by 
Loffler  and  Schiitz.  It  is  found  in  the  nasal  discharge  of 
glandered  animals.  In  appearance  the  bacillus  is  not  unlike 
B.  tuberculosis,  except  that  it  is  shorter  and  thicker.  The 
beading  of  the  bacillus  of  glanders,  like  that  in  tubercle, 
does  not  denote  spores.  B.  mallei  can  be  cultivated  on  the 
usual  media,  especially  on  glycerine  agar  and  potato.  On 
the  latter  medium  it  forms  a  very  characteristic  honey-like 
growth,  which  later  becomes  reddish-brown. 

In  the  horse  glanders  particularly  affects  the  nasal  mucous 
membrane,  forming  nodules  which  degenerate  and  emit  an 
offensive  discharge.  From  the  nose,  or  nasal  septum,  as  a 
centre,  the  disease  spreads  to  surrounding  parts.  It  may 
also  occur  as  nodules  in  and  under  the  skin,  when  it  is  known 
as  "  farcy."  Persons  attending  a  glandered  animal  may 
contract  the  disease,  often  by  direct  inoculation. 

Mallein  is  a  substance  analogous  to  tuberculin,  and  is 
made  by  growing  a  pure  culture  of  Bacillus  mallei  in  glycer- 
ine-veal broth  in  flat  flasks,  with  free  access  of  calcined  air. 
After  a  month's  growth  the  culture  is  sterilised,  filtered, 
concentrated,  and  mixed  with  an  equal  volume  of  a  .5  per 
cent,  solution  of  carbolic.  The  dose  is  i  cc.,  and  it  is  used, 
like  tuberculin,  for  diagnostic  purposes.  If  the  suspected 
animal  reacts  to  the  injection,  it  is  suffering  from  glanders. 


320  BACTERIA 

Reaction  is  judged  by  three  signs,  namely,  a  rise  of  tem- 
perature 2-3°  C.,  a  large  "  soup-plate  "  swelling  at  the  site 
of  inoculation,  and  an  enlargement  of  the  lymphatic 
glands. 

Swine  fever,  foot-and-mouth  disease,  chicken  cholera,  dysen- 
tery, rinderpest,  and  other  diseases  of  animals  have  micro- 
organisms intimately  related  to  them. 

There  is  a  group  of  diseases  due  to  the  presence  in  the 
blood  or  tissues  of  hcematozoa,  that  is,  protozoa  which  can 
live  and  perform  their  function  in  the  blood.  Amongst 
these  are  malaria,  sleeping  sickness,  and  other  tropical  dis- 
eases in  man,  and  surra  and  various  hsematozoa  in  horses, 
fish,  frogs,  or  rats, 

Malaria.  Although  a  Bacillus  malaria  has  been  de- 
scribed as  the  cause  of  this  disease,  it  is  now  almost  univer- 
sally supposed  that  the  true  cause  is  a  protozoan  parasite. 
In  1880  Laveran  first  described  this  organism,  and  the 
discovery  was  confirmed  by  Marchiafava,  Celli,  and  others. 
Laveran  claimed  that  it  occurred  in  four  different  forms 
during  the  progress  of  its  life-history : 

(a)  Spherical  or  Irregular  Bodies  attached  to  the  blood 
corpuscle,  or  free  in  the  blood  plasma.     They  are  a  little 
smaller  than  the  blood-cells,  and  may  or  may  not  contain 
pigment.     They  eventually  invade  the  corpuscles,  possess 
more  pigment,  and  lose  their  amoeboid  movement.     Within 
the  red  blood  corpuscles  they  increase  in  size  until  they  reach 
the  adult  stage. 

(b)  Segmentation  Forms,  often  assuming  a  rosette  shape, 
follow  next.     They  are  pigmented,  are  possibly  a  sporing 
stage,  and  are  finally  set  free  in  the  blood. 

(c)  The    Crescents,    or  Semilunar  Bodies,  are  free  in  the 
blood,  but  motionless.     They  are  colourless,  have  a  distinct 
membrane,  and  generally  show  a  little  pigment  about  the 
middle ;  they  taper  towards  the  poles.     They  appear  in  the 
blood  after  the  fever  has  existed  for  some  time,  occurring 


BACTERIA    AND   DISEASE  321 

chiefly,  sometimes  only,  in  the  quotidian  and  malignant 
types  of  malaria. 

(d )  The  Flagellated  Bodies  apparently  occur  only  in  the 
blood  outside  the  body.  They  are  extracorpuscular  bodies, 
and  possess  several  long  flagella,  and  are  therefore  actively 
motile.  They  are  derived  from  the  crescents  or  irregular 
intracorpuscular  bodies. 

What  is  the  precise  significance  of  these  various  forms 
and  modifications  of  them  is  not  at  present  known.  Pos- 
sibly the  semilune  is  a  resting  stage  inside  the  body,  and 
the  flagellated  body  another  similar  stage  outside.  At- 
tempts to  cultivate  the  parasite  outside  the  body  have  failed. 
There  is  a  good  deal  of  evidence  to  show  that  the  mosquito 
is  the  host  outside  the  human  body.  There  may  be  differ- 
ent forms  and  varieties  of  parasite,  if  not  actually  different 
species,  causing  the  diverse  forms  of  clinical  malaria. 

The  above  account  of  diseases  caused  by  bacteria  does 
not  profess  to  be  in  any  sense  exhaustive.  It  is  merely 
illustrative.  It  reveals  some  of  the  disease-producing  pow- 
ers of  micro-organisms.  There  are  a  large  number  of  other 
diseases  in  which  bacteria  have  been  found.  They  are  not 
the  causes,  but  only  accidentally  present  or  associated  with 
"  secondary  infection."  Variola  (small-pox),  scarlet  fever, 
and  measles  are  excellent  examples.  It  is  possible  that  the 
danger  at  the  present  time  is  rather  in  the  direction  of  sup- 
posing that  every  disease  will  readily  yield  its  secret  to  the 
bacteriologist.  Such,  of  course,  is  not  the  case.  Never- 
theless, as  in  the  past,  so  in  the  future,  constant  research 
and  patient  investigation  is  the  only  hope  we  have  for  the 
elucidation  of  truth  in  respect  to  the  causes  of  disease. 


CHAPTER  IX 

DISINFECTION 

THE  object  of  modern  bacteriology  is  not  merely  to  ac- 
cumulate tested  facts  of  knowledge,  nor  only  to  learn 
the  truth  respecting  the  biology  and  life-history  of  bacteria. 
These  are  most  important  things  from  a  scientific  point  of 
view.  But  they  are  also  a  means  to  an  end ;  that  end  is  the 
prevention  of  preventable  diseases  and  the  treatment  of  any 
departure  from  health.  In  a  science  not  a  quarter  of  a 
century  old  much  has  already  been  accomplished  in  this 
direction.  The  knowledge  acquired  of,  and  the  secrets 
learned  from,  these  tiny  vegetable  cells  which  have  such 
potentiality  for  good  or  evil  have  been,  in  some  degree, 
turned  against  them.  When  we  know  what  favours  their 
growth  and  vitality  and  virulence,  we  know  something  of 
the  physical  conditions  which  are  inimical  to  their  life; 
when  we  know  how  to  grow  them,  we  also  know  how  to 
kill  them. 

We  have  previously  made  a  cursory  examination  of  the 
methods  which  are  adopted  for  opposing  bacteria  and  their 
products  in  the  tissues  and  body  fluids.  We  must  now 
turn  to  consider  shortly  the  modes  which  may  be  adopted 
in  preventive  medicine  for  opposing  bacteria  outside  the 
body. 

It  will  be  clear  at  once  that  we  may  have  varying 
degrees  of  opposition  to  bacteria.  Some  substances  kill 
bacteria,  and  they  are  known  as  germicides ;  other  sub- 
stances prevent  their  development  and  resulting  septic 

322 


DISINFECTION  323 

action,  and  these  are  termed  antiseptics.  The  word  disin- 
fectant is  used  more  or  less  indiscriminately  to  cover  both 
these  terms.  A  deodorant  is,  of  course,  a  substance  remov- 
ing the  odour  of  evil-smelling  putrefactive  processes.  Here, 
then,  we  have  the  common  designations  of  substances  able 
to  act  injuriously  on  bacteria  and  their  products  outside,  or 
upon  the  surface  of,  the  body.  But  a  moment's  reflection 
will  bring  to  our  minds  two  facts  not  to  be  forgotten.  In 
the  first  place,  an  antiseptic  applied  in  very  strong  dose,  or 
for  an  extended  period,  may  act  as  a  germicide;  and,  vice 
versa,  a  germicide  in  too  weak  solution  to  act  as  such  may 
perform  only  the  function  of  an  antiseptic.  Moreover,  the 
action  of  these  disinfecting  substances  not  only  varies  ac- 
cording to  their  own  strength  and  mode  of  application,  but 
it  varies  also  according  to  the  specific  resistance  of  the  pro- 
toplasm of  the  bacteria  in  question.  Examples  of  the  latter 
are  abundant,  and  readers  who  have  only  assimilated  the 
simple  facts  set  forth  in  these  pages  are  aware  that  between 
the  bacillus  of  diphtheria  and  the  spores  of  anthrax  there 
is  an  enormous  difference  in  power  of  resistance.  In  the 
second  place,  reflection  will  enable  us  to  recall  what  has 
already  been  said,  when  discussing  the  requirements  neces- 
sary for  bacterial  growth,  respecting  the  physical  conditions 
injurious  to  development.  In  a  cold  temperature,  as  a  gen- 
eral rule,  bacteria  do  not  multiply  with  the  same  rapidity  as 
at  blood-heat.  Within  the  limits  of  a  moist  perimeter  the  air 
is,  to  all  intents  and  purposes,  germ-free.  Direct  sunlight 
has  a  definitely  germicidal  effect  in  the  course  of  time  upon 
some  of  the  most  virulent  bacteria  we  know.  Here,  then, 
are  three  examples  of  physical  agents — low  temperature, 
moist  perimeter,  sunlight — which,  if  strong  enough  in  de- 
gree, or  acting  for  a  long  enough  period  of  time,  become 
first  antiseptics  and  then  germicides.  Yet  for  a  limited 
period  they  have  no  injurious  effect  upon  bacteria.  These 
are  simple  points,  and  call  for  little  comment,  yet  the  pages 


324  BACTERIA 

of  medical  and  sanitary  journals  reveal  not  a  few  keen  con- 
troversies upon  the  injurious  action  of  certain  substances 
upon  certain  bacteria  owing  to  the  discrepancies,  of  neces- 
sity arising,  between  results  of  different  skilled  observers 
who  have  been  carrying  out  different  experiments  with  dif- 
ferent solutions  of  the  same  substance  upon  different  proto- 
plasms of  the  same  species  of  bacteria.  We  feel  no  doubt 
that  in  these  pioneering  researches  much  labour  has  been  to 
some  extent  misspent,  owing  to  the  neglect  of  a  common 
denominator.  Only  a  more  accurate  knowledge  of  bacteria 
or  a  recognised  standard  for  disinfecting  experiments  can 
ever  supply  such  common  denominator. 

Species  of  bacteria  for  comparative  observation-experi- 
ments upon  the  action  of  chemical  or  physical  agents  must 
be  not  only  the  same  species,  but  cultured  under  the  same 
conditions,  and  treated  by  the  agent  in  the  same  manner, 
otherwise  the  results  cannot  be  compared  upon  a  common 
platform,  or  with  any  hope  of  arriving  at  exactly  the  same 
conclusions. 

Sir  George  Buchanan  laid  down,  in  1884,  a  very  simple  and 
suitable  standard  of  what  true  disinfection  meant,  viz.,  the 
destruction  of  the  most  stable  known  infective  matter.  Such 
a  test  is  high  and  difficult  to  attain  unto ;  nevertheless,  it 
is  the  only  satisfactory  one.  Obviously  many  substances 
which  are  useful  antiseptics  in  practical  life  would  fall  far 
short  of  such  a  standard,  yet  for  true  and  complete  disin- 
fection such  an  ideal  is  the  only  adequate  one. 

Quite  recently  three  or  four  workers  at  Leipzig1  have 
drawn  up  simple  directions,  the  adoption  of  which  would 
considerably  assist  in  securing  a  common  standard  for  dis- 
infectant research.  They  are  as  follows : 

I.  In  all  comparative  observations  it  is  imperative  that 
molecularly  equivalent  quantities  of  the  reagents  should  be 
employed. 

1  Zeitschr.  f.  Hyg.  und  Inf.  Krank. ,  xxv. 


DISINFE  C  TION  325 

2.  The  bacteria  serving  as  test  objects  should  have  equal 
power  of  resistance. 

3.  The  numbers  of  bacteria  used  in  comparative  observa- 
tion should  be  approximately  equal. 

4.  The  disinfecting  solution  must  be  always  used  at  the 
same  temperature  in  comparative  experiments. 

5.  The  bacteria  must  be  brought  into  contact  with  the 
disinfectant  with  as  little  as  possible  of  the  nutrient  material 
carried  over.     (This  obviously  will  depend  upon  the  object 
of  the  research.) 

6.  After  having  been  exposed  to  the  disinfectant  for  a 
fixed  time,  they  should  be  freed  from  it  as  far  as  possible. 

7.  They  should  then  be  returned  in  equal  numbers  to  the 
respective  culture  medium  most  favourable  to  the  develop- 
ment of  each,  and  kept  at  the  same,  preferably  the  optimum, 
temperature  for  their  growth. 

8.  The  number  of  surviving  bacteria  capable  of  giving 
rise  to  colonies  in  solid  media  must  be  estimated  after  the 
lapse  of  equal  periods  of  time. 

We  may  now  turn  from  general  principles  to  mention 
shortly  some  of  the  commoner  methods  and  substances 
adopted  to  secure  efficient  disinfection.  They  are  all  divis- 
ible, according  to  Sir  George  Buchanan's  standard,  into  two 
groups: 

1.  Heat  in  various  forms; 

2.  Chemical  bodies  in  various  forms. 

It  should  at  the  outset  be  understood  that  we  desire  in 
practical  disinfection  to  inhibit  or  kill  micro-organisms  with- 
out injury  to,  or  destruction  of,  the  substance  harbouring 
the  germs  for  the  time  being.  If  this  latter  is  of  no  mo- 
ment, as  in  rags  or  carcasses,  burning  is  the  simplest  and 
most  thorough  treatment.  But  with  mattresses  and  bed- 
dings, bedclothes  and  garments,  as  well  as  with  the  human 
body,  it  is  obvious  that  something  short  of  burning  is 
required. 


326  BACTERIA 

i.  From  the  earliest  days  of  bacteriology  heat  has  held  a 
prominent  place  as  a  disinfector.  But  it  is  only  in  com- 
paratively recent  times  that  it  has  been  fully  established 
that  moist  heat  is  the  only  really  efficient  form  of  heat  dis- 
infection. Boiling  at  atmospheric  pressure  (100°  C.)  is  the 
oldest  form  of  moist  heat  disinfection,  and  because  of  the 
simplicity  of  its  application  it  has  gained  a  large  degree  of 
popularity.  But  it  must  not  be  forgotten  that  mere  boiling 
(100°  C.)  may  not  effectually  remove  the  spores  of  all  bacilli. 
Besides,  boiling  is  not  applicable  to  furniture,  mattresses, 
and  such-like  frequently  infected  objects.  For  many  of 
these  hot-air  ovens  were  used  in  the  early  days.  But  it  was 
found  that  such  disinfection  was  no  disinfection  at  all,  for 
not  only  did  it  leave  many  organisms  and  spores  untouched, 
but  the  degree  of  temperature  was  rarely,  if  ever,  uniform 
throughout  the  substance  being  treated. 

The  failures  following  in  the  track  of  these  methods  were 
an  indication  of  the  need  of  some  form  of  moist  heat,  viz., 
steam. 

Here  it  will  be  necessary  to  digress  for  a  moment  into 
some  of  the  characters  of  steam.  When  water  is  heated 
certain  molecular  changes  take  place,  and  at  a  certain  tem- 
perature (100°  C.,  212°  F.)  the  water  becomes  steam,  or 
vapour,  and  on  very  little  cooling  will  condense.  But  if  the 
vapour  is  heated,  it  will  become  practically  a  gas,  and  will 
not  condense  until  it  has  lost  the  whole  of  the  heat,  i.  e., 
the  heat  of  making  water  into  vapour  plus  the  heat  of 
making  vapour  into  gas.  A  gas  proper  is,  then,  the  vapour 
of  a  liquid  of  which  the  boiling-point  is  substantially  below 
its  actual  temperature.  But  we  know  that  the  temperature 
at  which  it  boils  depends  upon  the  pressure  to  which  it 
is  subjected  (Regnault's  law).  Hence  in  reality  "  steam 
at  any  temperature  whatever  may  be  a  vapour  proper, 
provided  the  pressure  is  such  as  prevents  the  liquid  from 
boiling  below  that  temperature."  In  such  a  condition 


DISINFECTION  327 

of  vapour  it  is  termed  saturated  steam.  But  if  it  is  at  that 
same  pressure  further  heated,  it  becomes  practically  a  gas, 
and  is  called  superheated  steam.  The  former  can  condense 
without  cooling;  the  latter  cannot  so  condense  at  the  same 
pressure.  Saturated  steam  condenses  immediately  it  meets 
the  object  to  be  disinfected,  and  gives  out  its  latent  heat ; 
superheated  steam  acts  by  conduction,  and  not  uniformly 
throughout  the  object.  Its  advantage  is  that  it  dries  moist- 
ened objects.  As  a  disinfecting  power,  superheated  steam 
is  much  less  than  saturated  steam.  There  is  one  further 
term  which  must  be  defined,  namely,  current  steam.  This 
is  steam  escaping  from  a  disinfector  as  fast  as  it  is  admitted, 
and  may  be  at  atmospheric  or  higher  temperatures.  The 
disinfecting  temperature  which  is  now  used  as  a  standard  is 
an  exposure  to  saturated  steam  of  nf  C.  for  fifteen  minutes. 
A  number  of  different  kinds  of  apparatus  have  been  in- 
vented to  facilitate  disinfection  to  this  standard  on  a  large 
scale.  Most  sanitary  authorities  of  importance  are  now 
supplied  with  some  form  of  steam  disinfector,  though  many 
are  unable  to  go  to  the  expense  of  high-pressure  disinfectors. 
Professor  Delepine  has  pointed  out '  that  a  current  of  steam 
at  low  pressure  may  completely  disinfect.  Whilst  such 
simple  current-steam  machines  have  thus  been  demonstrated 
as  efficient  bactericides,  for  all  practical  purposes  it  is  im- 
portant to  have  disinfectors  capable  of  giving  temperatures 
considerably  above  100°  C.,  of  simple  construction,  having 
steam  power  of  uniform  temperature  and  rapid  penetration, 
and  containing,  when  in  action,  a  minimum  of  superheated 
steam.  In  addition  to  these  characters  of  a  first-rate  steam 
disinfector,  two  other  important  points  should  be  borne  in 
mind,  namely,  the  air  must  be  completely  ejected  from  the 
disinfection  chamber  before  the  results  due  to  steam  are 
obtained,  and  some  sort  of  automatic  index  giving  a  record 
of  each  disinfection  is  indispensable. 

1  Journal  of  State  Medicine,  December,  1897,  p.  561. 


328  BACTERIA 

We  may  turn  from  these  general  principles  to  mention 
shortly  some  of  the  types  of  steam  disinfectors  most  com- 
monly in  use.  They  are  four,  namely,  the  Washington 
Lyon,  the  Equifex  (Defries),  the  Thresh,  and  the  Reck. 

Washington  Lyon  s  apparatus  consists  of  an  elongated 
boiler  having  double  walls,  with  a  door  at  each  end.  The 
body  of  the  apparatus  is  jacketed.  The  whole  is  large 
enough  to  admit  of  bedding  and  mattresses,  and  generally 
is  so  arranged  that  one  end  opens  into  one  room,  and  the 
other  end  opens  into  another  room.  This  convenient  posi- 
tion admits  of  inserting  infected  articles  from  one  room  and 
receiving  them  disinfected  into  the  other  room.  Possible 
reinfection  is  thereby  prevented.  Steam  is  admitted  into 
the  jacket  at  a  pressure  of  between  twenty  and  twenty-five 
pounds,  and  is  generally  twenty  pounds  in  the  interior  of 
the  cylinder.  At  the  end  of  the  operation  a  partial  vacuum 
is  created,  by  which  means  much  of  the  moisture  on  the 
articles  may  be  removed.  In  some  cases  a  current  of  warm 
air  is  admitted  before  disinfection  in  order  to  diminish  the 
extent  of  condensation. 

The  Equifex  (Defries)  contains  no  steam  jacket,  but  coils 
of  pipes  are  placed  at  the  top  and  bottom  of  the  apparatus, 
with  the  object  of  imparting  to  the  steam  as  much  heat  as 
is  lost  by  radiation  through  the  walls  of  the  disinfecting 
chamber,  and  at  the  same  time  of  preventing  undue  con- 
densation. The  air  is  first  removed  by  a  preliminary  cur- 
rent of  steam,  after  which  steam  at  a  pressure  of  ten  pounds 
is  intermittently  introduced  and  allowed  to  escape.  The 
object  of  this  proceeding  is  to  remove  air  from  the  pores  of 
the  articles  to  be  disinfected  by  the  sudden  expansion  of  the 
film  of  water  previously  condensed  on  their  surface.  The 
apparatus  introduced  by  Dr.  Thresh  was  constructed  with  a 
view  of  overcoming  the  objection  to  some  of  the  other 
machines  that  bulky  articles  retained  a  large  percentage  of 
moisture,  thus  necessitating  the  use  of  some  additional 


DISINFECTION  329 

drying  apparatus.  A  central  chamber  receives  the  articles 
to  be  disinfected,  and  is  surrounded  by  a  boiler  containing 
a  solution  of  calcium  chloride  at  a  temperature  of  225°  F. 
This  is  heated  by  a  small  furnace,  and  the  steam  given  off 
(218-300°  F.)  is  conducted  into  the  central  chamber.  The 
steam  is  not  confined  under  any  pressure  except  that  of  the 
atmosphere.  When  the  steam  has  passed  for  a  sufficient 
length  of  time,  it  is  readily  diverted  into  the  open  air.  Hot 
air  is  now  introduced,  and  at  the  expiration  of  an  hour  the 
articles  may  be  taken  out  disinfected  and  as  dry  as  they  were 
when  inserted.  The  apparatus  is  comparatively  inexpens- 
ive, and  not  of  a  complicated  nature.  The  current  steam 
is  saturated,  and  at  a  temperature  a  few  degrees  above  the 
boiling-point.  Many  experiments  have  been  performed 
with  this  apparatus,  and  there  is  now  a  large  amount  of 
evidence  in  favour  of  it  and  current  steam  disinfection. 

Reck' s  apparatus  is  another  kind  of  saturated  steam  disin- 
fector,  which  resembles  the  Equifex,  but  differs  from  it  in 
employing  steam  as  a  current. 

It  is  probable  that  many  other  forms  of  steam  disinfector 
will  be  invented,  and  each  will  have  its  enthusiastic  sup- 
porters. Even  at  the  time  of  writing  some  excellent  results 
are  announced  from  America. 

2.  The  effects  of  chemical  substances  as  solutions,  or  in 
spray  form,  upon  bacteria  have  been  observed  from  the 
earliest  days  of  bacteriology.  To  some  decomposing  matter 
or  solution  a  disinfectant  was  added  and  sub-cultures  made. 
If  bacteria  continued  to  develop,  the  disinfection  had  not 
been  efficient;  if,  on  the  other  hand,  the  sub-culture  re- 
mained sterile,  disinfection  had  been  complete.  From  such 
rough-and-ready  methods  large  deductions  were  drawn,  and 
it  is  hardly  too  much  to  say  that  no  branch  of  bacteriology 
contains  such  a  vast  mass  of  unassimilated  and  unassimilable 
statements  as  that  relating  to  research  into  disinfectants. 
Most  of  the  tabulated  and  recorded  results  are  conspicuous 


330  BACTERIA 

in  having  no  standard  as  regards  bacterial  growth.     Yet 
without  such  a  standard  results  are  not  comparable. 

Silk  threads,  impregnated  with  anthrax  spores,  were 
placed  in  bottles  containing  carbolic  acid  of  various 
strengths,  and  at  stated  periods  threads  were  removed  and 
placed  in  nutrient  media,  and  development  or  otherwise 
observed.  But,  as  Professor  Crookshank  l  has  pointed  out, 
this  method  is  fallacious,  the  thread  being  still  wet  with  the 
solution  when  transferred  to  the  medium,  and  thus  modi- 
fied in  culture,  possibly  even  inhibited  altogether.  It  is 
unnecessary  for  us  here  to  discuss  every  mode  adopted  by 
investigators  in  similar  researches.  We  may  just  mention 
that  the  most  approved  methods  at  the  present  time  are 
based  upon  two  simple  plans  of  exposure.  In  one  we  use  a 
known  volume  of  recent  broth  culture  of  an  organism  grown 
under  specified  conditions.  To  this  is  added  a  measured 
quantity  of  the  antiseptic.  At  stated  periods  loopfuls  of 
the  broth  and  antiseptic  mixture  are  sub-cultured  in  fresh- 
sterilised  broth,  and  resulting  development  or  otherwise 
closely  observed.  The  other  method  is  practicable  when 
we  are  dealing  with  volatile  bodies.  In  such  cases  a  stand- 
ard culture  is  made  of  the  organism  in  broth  at  a  standard 
temperature.  Into  this  are  dipped  small  strips  of  sterilised 
linen.  When  thoroughly  impregnated  these  are  removed 
from  the  broth  and  subsequently  dried  over  sulphuric  acid 
in  a  vacuum  at  38°  C.  These  may  now  be  exposed  for  a 
longer  or  shorter  period  to  the  fumes  of  the  antiseptic  in 
question,  and  broth  cultures  made  at  the  end  of  the  expos- 
ure. It  is  obvious  that  a  very  large  number  of  modifications 
are  possible  of  these  two  simple  devices  for  testing  the  bac- 
tericidal power  of  chemical  substances.  It  should  be  re- 
membered that  here,  perhaps,  more  than  anywhere  else  in 
bacteriological  research,  careful  control  experiments  are 
absolutely  necessary. 

1  Bacteriology  and  Infective  Diseases,  p.  35. 


DISINFECTION 


331 


Mineral  acids  (nitric,  hydrochloric,  sulphuric),  especially 
concentrated,  are  all  germicides. 

The  halogens — chlorine,  bromine,  iodine,  and  fluorine — 
are,  all  four,  disinfectants,  but  not  used  in  practice.  They 
are  named  in  their  order  of  power  as  such. 

A  number  of  separate  bodies,  such  as  chloroform  and 
iodoform,  have  been  much  advocated  as  antiseptics.  The 
cost  of  the  former  and  odour  of  the  latter  have,  however, 
greatly  militated  against  their  general  adoption. 

Chloride  of  lime  is  a  powerful  disinfectant.  Professor 
Sheridan  Delepine  and  Dr.  Arthur  Ransome  have  demon- 
strated its  germicidal  effect  as  a  solution  applied  directly  to 
the  walls  of  rooms  inhabited  by  tuberculous  patients.1  It 
may  also  be  used  in  solid  form  for  dusting  decomposing 
matter. 

Mercuric  chloride  (corrosive  sublimate)  has  been  an  ac- 
cepted germicide  for  some  time.  But  the  experiments  of 
Behring,  Crookshank,  and  others  have  proved  that  the 
weaker  solutions  cannot  be  relied  upon.  This  is,  in  part, 
due  to  the  fact  that  it  forms  in  albuminous  liquids  an  albu- 
minate  of  mercury  which  is  inactive.  Dilute  solutions  have 
the  further  disadvantage  of  being  unstable.  Various  au- 
thorities recommend  a  solution  of  1-500  as  a  germicide,  and 
much  weaker  solutions  are,  of  course,  antiseptic.  An  ounce 
each  of  corrosive  sublimate  and  hydrochloric  acid  in  three 
gallons  of  water  makes  an  efficient  disinfectant. 

Potassium  permanganate  is,  of  course,  the  chief  substance 
in  Condy's  fluid,  as  zinc  chloride  is  in  Burnett's  disinfecting 
fluid.  A  5  per  cent,  of  the  former  and  a  2J-  per  cent,  of  the 
latter  are  germicidal. 

Boracic  acid  is  one  of  the  most  useful  antiseptics  with 
which  to  wash  sore  eyes,  or  preserve  tinned  foods  or  milk.  It 
is  not  a  strong  germicide,  but  an  unirritating  and  effective 
wash.  Many  cases  of  its  addition  to  milk  have  found  their 

1  British  Medical  Journal,  1895  (February),  p.  353. 


332  BACTERIA 

way  into  the  law  courts,  owing  to  cumulative  poisoning,  and 
it  should  only  be  used  with  the  very  greatest  care  as  a  food 
preservative. 

Carbolic  acid  has  come  into  prominence  as  an  antiseptic 
since  its  adoption  by  Lister  in  antiseptic  surgery.  It  is 
cheap,  volatile,  and  effective.  One  part  in  400  is  antiseptic, 
and  i  in  20  germicidal.  As  a  wash  for  the  hands  the  former 
is  used,  and  a  weaker  solution  for  the  body  generally.  Car- 
bolic soap  and  similar  toilet  combinations  are  now  very 
common.  At  one  time  it  appeared  as  if  corrosive  sublimate 
would  oust  carbolic  from  the  first  place  as  an  antiseptic 
solution,  but  a  large  number  of  experiments  have  confirmed 
opinion  in  favour  of  carbolic.  Professor  Crookshank  found 
that  carbolic  acid,  I  in  40,  acting  for  only  one  minute  is 
sufficient  to  destroy  Streptococcus  pyogenes,  S.  erysipelatis, 
and  Staphylococcus  pyogenes  aureus,  and  in  the  strength  of  I 
in  20  carbolic  acid  completely  sterilised  tubercular  sputum 
when  shaken  up  with  it  for  one  minute. 

Creosol,  a  member  of  the  phenol  series,  is  a  good  disin- 
fectant, and  the  active  element  in  lysol,  Jeye's  fluid,  creo- 
line,  izal,  and  creosote. 

Sulphurous  acid  is  one  of  the  commonest  disinfectants 
employed  for  fumigation — the  old  orthodox  method  of  dis- 
infecting a  room  in  which  a  case  of  infective  disease  has 
been  nursed.  It  is  evolved,  of  course,  by  burning  sulphur. 
For  each  thousand  cubic  feet  from  one  to  five  pounds  of 
sulphur  is  used,  and  the  walls  may  be  washed  with  car- 
bolic acid.  Dr.  Kenwood  carried  out  some  experiments 
in  1896 1  which  appear  to  support  the  disinfecting  power 
of  sulphur  fumes.  He  found  that  the  Bacillus  diphtheria 
was  not  killed,  though  markedly  inhibited,  when  the  sul- 
phurous gas  (SO3)  did  not  much  exceed  .25  per  cent. 
But  the  bacillus  was  killed  where  the  sulphur  fumes  ex- 
ceeded .5  per  cent.  Both  these  results  had  reference  to  the 

1  British  Medical  Journal,  1896  (August),  p.  439. 


DISINFECTION 


333 


SO2  in  the  air  in  the  centre  of  the  room  at  a  height  of  four 
feet,  and  after  the  lapse  of  four  hours.  There  can  be  little 
doubt  that  fuming  a  sealed-up  room  with  sulphur  fumes  in 
a  moist  atmosphere,  and  leaving  it  thus  for  twenty-four 
hours,  is  generally,  if  not  always,  efficient  disinfection.  It 
will  kill  the  bacillus  of  diphtheria,  though  not  always  more 
resistant  germs.  Moreover,  its  simplicity  of  adoption  is 
greatly  in  its  favour.  Anyone  can  readily  apply  it  by  pur- 
chasing a  few  pounds  weight  of  ordinary  roll  sulphur  and 
burning  this  in  a  saucer  in  the  middle  of  a  room  which  has 
had  all  its  crevices  and  cracks  in  windows  and  walls  blocked 
up  with  pasted  paper.  Nitrous  fumes  may  also  be  used  in 
this  way. 

Recently  formalin  has  come  much  in  favour  as  a  room 
disinfectant.  Formalin  is  a  40  per  cent,  solution  of  form- 
aldehyde in  water,  a  gas  discovered  by  Hofmann  in  1869. 
This  gas  is  a  product  of  imperfect  oxidation  of  methyl 
alcohol,  and  may  be  obtained  by  passing  vapour  of  methyl 
alcohol,  mixed  with  air,  over  a  glowing  platinum  wire  or 
other  heated  metals,  such  as  copper  and  silver.  It  is  the 
simplest  of  a  series  of  aldehydes,  the  highest  of  which  is 
palmitic  aldehyde.  Its  formula  is  CH3O,  and  it  is  a  colour- 
less gas  with  a  pungent  odour,  and  having  penetrating  and 
irritating  properties,  particularly  affecting  the  nasal  mucous 
membrane  and  the  eyes  of  those  working  with  it.  It  is 
readily  soluble  in  water,  and  in  the  air  oxidises  into  formic 
acid  (CH2O3).  This  latter  substance  occurs  in  the  stings 
of  bees,  wasps,  nettles,  and  various  poisonous  animal  secre- 
tions. Formalin  is  a  strong  bactericide  even  in  dilute 
solutions,  and,  of  course,  volatile.  A  solution  of  I  to 
10,000  is  said  to  be  able  to  destroy  the  bacilli  of  typhoid, 
cholera,  and  anthrax.  A  teaspoonful  to  ten  gallons  of  milk 
is  said  to  retard  souring.  When  formalin  is  evaporated 
down,  a  white  residue  is  left  known  as  paraform.  In  lozenge 
form  this  latter  body  is  used  by  combustion  of  methylated 


334  BACTERIA 

spirit  to  produce  the  gas.  Hence  we  have  three  common 
forms  of  the  same  thing — formalin,  formic  aldehyde,  para- 
form — each  of  which  yields  formic  acid,  and  thus  disinfects. 
The  vapour  cannot  in  practice  be  generated  from  the  forma- 
lin as  readily  as  from  the  paraform. 

By  a  variety  of  ingenious  arrangements  formic  aldehyde 
has  been  tested  by  a  large  number  of  observers  during  the 
last  two  or  three  years.  We  may  refer  to  three  modes  of 
application.  I.  The  sprayer  (Equifex  apparatus)  produces 
a  mixture  of  air  and  solution  for  spraying  walls,  ceilings, 
floors,  and  sometimes  garments.  2.  The  autoclave  (Tr\\\a.\.'s 
apparatus).  In  this  a  mixture  of  a  30-40  per  cent,  watery 
solution  of  formaldehyde  and  calcium  chloride  (4-5  per 
cent.)  is  heated  under  a  pressure  of  three  or  four  atmos- 
pheres, and  the  almost  pure,  dry  gas  is  conducted  through 
a  tube  passing  through  the  keyhole  of  the  door  into  the 
sealed-up  room.  3.  The  paraform  lamp  (the  Alformant). 
The  principle  of  this  lamp  is  that  the  hot,  moist  products 
from  the  combustion  of  methylated  spirit  act  upon  the  para- 
form tablets,  converting  them  into  gas.  Most  of  the  con- 
clusions derived  from  experiments  with  these  three  different 
forms  of  apparatus  are  the  same.  It  is  agreed  that  the  gas 
is  harmless  to  colours  and  metal  and  polished  wood.  The 
vapour  acts  best  in  a  warm  atmosphere.  As  for  its  action 
on  bacteria,  it  compares  favourably  with  any  other  disinfect- 
ant. In  i  per  cent,  solution  formalin  destroys  non-spore- 
bearing  bacteria  in  thirty  to  sixty  minutes. 

Many  observers  have  decried  formaldehyde  on  account  of 
its  professed  lack  of  penetrating  power.  Professor  Delepine, 
however,  states  '  that  it  possesses  "  penetration  powers  prob- 
ably greater  than  those  of  most  other  active  gaseous  disin- 
fectants. Bacillus  colt,  B.  tuberculosis,  B.  pyocyaneus,  and 
Staph.  pyogenes  aureus  were  killed  in  dry  or  moist  state, 
even  when  protected  by  three  layers  of  filter  paper."  In 

1  Journal  of  State  Medicine,  1898  (November),  p.  541. 


DISINFECTION  335 

Professor  Delepine's  opinion,  the  vapours  of  phenol,  izal,, 
dry  chlorine,  and  sulphurous  acid  have,  under  the  same 
conditions,  given  inferior  results. 

We  may  now  shortly  summarise  the  foregoing  facts  re- 
specting antiseptics  and  disinfection  in  the  simplest  terms 
possible  to  afford  facility  to  the  uninitiated  in  practical 
application : 

To  disinfect  a  room,  seal  up  cracks  and  crevices,  and  burn 
at  least  one  pound  of  roll  sulphur  for  every  1000  cubic  feet 
of  space.1  Many  authorities  recommend  four  or  five  pounds 
of  sulphur  to  the  same  space.  Let  the  room  remain  sealed 
up  for  twenty-four  hours. 

To  disinfect  walls,  wash  with  chloride  of  lime  solution 
(i-ioo)  or  carbolic  acid  (1-40).  This  latter  solution  may  be 
used  to  wipe  down  furniture.  Either  or  both  may  be  used 
after  sulphur  fuming.  Formic  aldehyde  may  also  be  used 
by  lamp  or  autoclave. 

To  disinfect  bedding,  etc. ,  the  steam  sterilisation  secured 
in  a  Thresh,  Equifex,  or  Lyon  apparatus  is  the  best.  Rags 
and  infected  clothing,  unless  valuable,  should  be  burnt. 

To  disinfect  garments  and  wearing  apparel,  they  should  be 
washed  in  a  disinfectant  solution,  or  fumed  with  formic 
aldehyde. 

To  disinfect  excreta  or  putrefying  solutions,  enough  disin- 
fectant should  be  added  to  produce  in  the  solution  or  matter 
being  disinfected  the  percentage  of  disinfectant  necessary  to 
act  as  such.  Adding  a  small  quantity  of  antiseptic  to  a 
large  volume  of  fluid  or  solid  is  as  useless  as  pouring  a  small 
quantity  of  antiseptic  down  a  sewer  with  the  idea  that  such 
treatment  will  disinfect  the  sewage.  The  mixture  of  the 
disinfectant  with  the  matter  to  be  disinfected  must  contain 
the  standard  percentage  for  disinfection.  Chloride  of  lime 
is  a  common  substance  for  use  in  this  way.  Potassium 

1  The  measurement  of  cubic  space  is  of  course  made  by  multiplying  together 
in  feet  the  length,  breadth,  and  height  of  a  room. 


336  BACTERIA 

permanganate  (i-ioo)  and  carbolic  (i-ioo),  and  many  manu- 
factured bodies  containing  them,  are  also  widely  used.  Drs. 
Hill  and  Abram  recommend  J  that  the  excreta  and  disinfect- 
ant be  thoroughly  mixed  and  stand  for  at  least  half  an  hour. 
For  various  reasons  they  particularly  advise  chinosol  as  the 
most  convenient  disinfectant  for  this  specific  purpose. 

Antiseptics  for  wounds.  Carbolic  acid  (1-40)  or  corrosive 
sublimate  (i-iooo)  are  commonly  used  in  surgical  practice. 
Boracic  acid  is  one  of  the  most  unirritating  antiseptics  which 
are  known.  It  may  be  used  in  saturated  watery  solution 
(1-30)  or  dusted  on  copiously  as  fine  powder.  It  is  espe- 
cially applicable  in  open  wounds,  and  as  an  eye-wash. 

To  disinfect  hands  and  arms.  Operating  surgeons  are 
those  to  whom  it  is  a  most  urgent  necessity  to  cleanse  hands 
and  arms  antiseptically.  Carbolic  acid  (1-20,  or  1-40)  is 
used  for  this  purpose. 

It  is  hardly  necessary  to  add  that  in  a  case  of  infectious 
disease  occurring  in  a  household  many  of  these  modes  of 
application,  perhaps  all  of  them,  must  be  adopted.  Form- 
alin is  probably  the  best  gaseous  disinfectant  which  we 
have,  but  its  use  does  not,  and  should  notv  preclude  the 
simultaneous  adoption  of  other  methods. 

1  British  Medical  Journal,  1898  (April),  p.  1013. 


APPENDIX 

IT  is  proposed  to  add  one  or  two  notes  on  certain  technical 
points  in  bacteriological  work,  with  a  view  to  assisting  those 
medical  men  not  able  to  obtain  the  advantages  of  a  well-equipped 
laboratory,    and   yet   desirous  of  occasionally   attempting  some 
practical  bacteriology. 

i.  General  Examination.  All  fluids  may  be  examined  for  bac- 
teria in  two  chief  ways: 

(a)  A  small  quantity  may  be  placed  on  a  cover-glass  or  slide, 
dried  over  a  lamp  or  bunsen  flame,  and  stained  with  aniline  dyes 
for  a  few  minutes.  It  is  then  ready  for  microscopic  examination. 
It  is  obvious  that  the  result  will  generally  be  a  mixture  of  bacteria, 
for  which  differentiating  stains  may  be  used  (Gram,  Ziehl-Neel- 
sen,  etc.).  * 

(£)  A  minute  drop  of  the  suspected  fluid  may  be  added  to 
various  fluid  media  (broth,  liquefied  gelatine,  etc.)  and  then 
plated  out  upon  small  sterilised  sheets  of  glass.  In  the  course 
of  two  or  three  days  the  contained  bacteria  will  reveal  themselves 
in  characteristic  colonies,  which  may  be  examined,  and  if  possible 
sub-cultured,  and  carefully  studied. 

Double- Staining  Methods.  These  are  various,  and  are  used 
when  it  is  desired  to  stain  the  bacteria  themselves  one  colour, 
and  the  matrix  or  ground  substance  in  which  they  are  situated 
another  colour.  Three  of  the  commoner  methods  are  those  of 
Ehrlich,  Neelsen,  and  Gram.  They  are  as  follows: 

Ehr lie fi  s  Method.  "  Five  parts  of  aniline  oil  are  shaken  up 
with  100  parts  distilled  water,  and  the  emulsion  filtered  through 
moistened  filter  paper.  A  saturated  alcoholic  solution  of  fuchsine, 
methyl-violet,  or  gentian-violet  is  added  to  the  filtrate  in  a  watch- 

22 

337 


338  APPENDIX 

glass,  drop  by  drop,  until  precipitation  commences.  Cover-glass 
preparations  are  floated  in  this  mixture  for  fifteen  to  thirty  minutes, 
then  washed  for  a  few  seconds  in  dilute  nitric  acid  (one  part  nitric 
acid  to  two  of  water),  and  then  rinsed  in  distilled  water.  The 
stain  is  removed  from  everything  except  the  bacilli;  but  the 
ground  substance  can  be  after-stained  brown  if  the  bacilli  are 
violet,  or  blue  if  they  have  been  stained  red"  (Crookshank, 
Bacteriology  and  Infective  Diseases,  p.  89). 

Gram 's  Method.  The  primary  stain  in  this  method  is  a  solu- 
tion of  aniline  gentian-violet  (saturated  alcoholic  solution  of 
gentian-violet  30  cc.,  aniline  water  100  cc.),  which  stains  both 
ground  substance  and  bacteria  in  purple.  The  preparation  is 
next  immersed  in  the  following  solution  for  half  a  minute  or  a  little 
more: 

Iodine i  part 

Potassium  iodide 2  parts 

Distilled  water 300  parts 

In  this  short  space  of  time  the  iodine  solution  acts  as  a  mord- 
ant of  the  purple  colour  in  the  bacteria,  but  not  in  the  ground 
substance.  Hence,  if  the  preparation  be  now  (when  it  has  as- 
sumed a  brown  colour)  washed  in  alcohol  (methylated  spirit),  the 
ground  substance  slowly  loses  its  colour  and  becomes  clear.  But 
the  bacteria  retain  their  colour,  and  thus  stand  out  in  a  well- 
defined  manner.  Cover-glass  preparations  decolourise  more 
quickly  than  sections  of  hardened  tissue,  and  they  should  only  be 
left  in  the  methylated  spirit  until  no  more  colour  comes  away. 
The  preparation  may  now  be  washed  in  water,  dried,  and 
mounted  for  microscopic  examination,  or  it  may  be  double- 
stained,  that  is,  immersed  in  some  contrast  colour  which  will 
lightly  stain  the  ground  substance.  Eosin  or  Bismarck  brown 
are  commonly  used  for  this  purpose.  The  former  is  applied  for 
a  minute  or  two,  the  latter  for  five  minutes,  after  which  the 
specimen  is  passed  through  methylated  spirit  (and  preferably 
xylol  also)  and  mounted.  The  result  is  that  the  bacteria  appear 
in  a  dark  purple  colour  on  a  background  of  faint  pink  or  brown. 
Carbol-thionine  blue,  picro-carmine,  and  other  stains  are  occa- 


APPENDIX 


339 


sionally  used  in  place  of  the  aniline  gentian- violet,  and  there  are 
other  slight  modifications  of  the  method. 

Ziehl-Neelsen  Method.  Here  the  primary  stain  is  a  solution  of 
carbol-fuchsin : 

Fuchsin i  part 

Absolute  alcohol 10  parts 

5  per  cent,  aqueous  solution  of  carbolic  acid. . .    100  parts 

It  is  best  to  heat  the  dye  in  a  sand-bath,  in  order  to  distribute 
the  heat  evenly.  The  various  stages  in  the  staining  process  are 
as  follows:  (a)  The  cover-glass  with  the  dried  film  upon  it  is 
immersed  in  the  hot  stain  for  one  to  three  minutes.  (<£)  Remove 
the  cover-glass  from  the  carbol-fuchsin,  and  place  it  in  a  capsule 
containing  a  25  percent,  solution  of  sulphuric  acid  to  decolourise 
it.  Here  its  redness  is  changed  into  a  slate-grey  colour,  (c) 
Wash  in  water,  and  alternately  in  the  acid  and  water,  until  it  is 
of  a  faint  pink  colour,  (d)  Now  place  the  cover-glass  for  a 
minute  or  two  in  a  saturated  aqueous  solution  of  methylene-blue, 
which  will  counter-stain  the  decolourised  ground  substance  blue. 
(e)  Wash  in  water.  (/)  Dehydrate  by  rinsing  in  methylated  spirit, 
dry,  and  mount.  A  pure  culture  of  bacteria  will  not  necessarily 
require  the  counter-stain  (methylene-blue).  Sections  of  tissue 
may  require  twenty  to  thirty  minutes  in  the  primary  stain  (carbol- 
fuchsin).  This  stain  is  used  for  tubercle  and  leprosy.  With  a 
little  practice  the  staining  of  the  bacillus  of  tubercle  when  present 
in  pus  or  sputum  becomes  a  very  simple  and  accurate  method  of 
diagnosis.  A  small  particle  of  sputum  or  pus  is  placed  between 
two  clean  cover-glasses  and  thus  pressed  between  the  thumb  and 
finger  into  a  thin  film.  This  is  readily  dried  and  stained  as 
above,  the  bacillus  of  tubercle  appearing  as  a  delicately-beaded 
red  rod  with  a  background  of  blue. 

Bacteriological  Diagnosis.  The  following  points  must  be  ascer- ' 
tained  in  order  to  identify  any  particular  micro-organism : 

(1)  Its  morphology,  bacillus,  coccus,  spirillum,  etc.;  the  pres- 
ence or  absence  of  involution  forms. 

(2)  Motility  by  the  unstained  cover-glass  preparation  ("  hang- 
ing drop  ");  note  presence  of  flagella. 


340 


APPENDIX 


(3)  Presence  of  spores,  their  appearance  and  position. 

(4)  Whether  or  not  the  organism  stains  with  Gram's  method. 

(5)  The  character  of  the  growth  upon  various  media  (gelatine, 
agar,  milk,  potato,  broth) ;  the  presence  or  absence  of  liquefaction 
in  the  gelatine  culture;  its  power  of  producing  acid,  gas,  or  indol. 

(6)  Whether  it  is  aerobic  or  anaerobic. 

(7)  Its  colour  in  cultivation. 

(8)  If  it  is  a  disease-producing  organism  under  examination, 
its  effect  upon  the  animal  tissues  and  the  course  of  the  disease 
should  be  observed. 

There  are  other  points  of  importance,  but  the  above  are  essen- 
tial to  a  right  conclusion. 

Diagnosis  in  Special  Diseases  : 

(1)  Diphtheria.     This  disease  may  be  bacteriologically  diag- 
nosed with  a  minimum  of  apparatus  and  equipment.     By  means 
of  a  swab  a  rubbing  from  a  suspected  throat  is  readily  obtained. 
This  may  be  examined  by  the  microscope,  or  sub-cultured  on 
favourable  medium.     Blood  serum  is  perhaps  the  best,  but,  as 
Hewlett  remarks,  "If  no  serum  tubes  can  be  had,  an  egg  may 
be  used.     It  is  boiled  hard,  the  shell  chipped  away  from  one  end 
with  a  knife  sterilised  by  heating,  and  the  inoculation  made  on 
the  exposed  white  surface;  the  egg  is  then  placed,  inoculated  end 
down,  in  a  wine-glass  of  such  a  size  that  it  rests  on  the  rim  and 
does  not  touch  the  bottom.     A  few  drops  of  water  may  with  ad- 
vantage be  put  at  the  bottom  of  the  glass  to  keep  the  egg-white 
moist.     The  preparation  is  kept  in  a  warm  place  for  twenty-four 
to  forty-eight  hours  and  then  examined."     The  examination,  of 
course,  consists  in  staining  and  preparing  for  the  microscope  and 
observing  the  form,  arrangement,  and  characters  of  the  organism 
or  organisms  present.     A  small  piece  of  the  membrane  may  be 
detached,  washed  in  water,  and  stained  for  the  bacilli. 

(2)  Tubercle  (Ziehl-Neelsen's  stain,  vide  supra). 

(3)  Typhoid  (Enteric  Fever). 

Widal' s  Reaction.  This  diagnostic  test  depends  upon  the  effect 
which  the  blood  of  a  person  suffering  from  typhoid  fever  has 
upon  the  Bacillus  typhosus.  The  effect  is  twofold.  In  the  first 


APPENDIX  34 1 

place,  the  actively  motile  B.  typhosus  becomes  immotile;  and 
secondly,  there  is  an  agglutination,  or  grouping  together  in 
colonies,  of  the  B.  typhosus.  Neither  of  these  features  occurs  if 
healthy  human  blood  is  brought  into  contact  with  a  culture  of  the 
typhoid  bacillus.  There  are  various  ways  in  which  this  "  serum 
diagnosis "  can  be  carried  out.  The  simplest  and  quickest 
method  is  as  follows:  To  ten  drops  of  a  twenty-four  or  forty- 
eight-hours-old  neutral  broth  culture  of  the  typhoid  bacillus  one 
drop  of  the  blood  serum  to  be  tested  is  added.  The  serum  and 
culture  are  rapidly  mixed  in  the  trough  of  a  hollow  ground  slide 
(such  as  is  used  for  the  "  hanging  drop  "),  and  a  single  drop  is 
taken,  placed  upon  an  ordinary  clean  slide,  and  a  cover-glass 
superimposed.  The  positive  reaction  of  agglutination  and  im- 
motility,  if  the  blood  comes  from  a  case  of  typhoid  fever,  will 
probably  appear  within  fifteen  or  twenty  minutes.  The  fluid 
culture  of  typhoid  may  be  taken  from  an  agar  culture  as  well  as 
from  broth.  In  both  cases  it  may  be  desirable  to  filter  through 
ordinary  filter  paper  to  remove  any  normally  agglutinated  masses 
of  bacilli  before  commencing  the  test. 

In  his  first  experiments  Widal  used  a  test-tube  in  the  following 
manner:  The  blood  to  be  tested  is  diluted  by  one  part  of  it  being 
added  to  fifteen  parts  of  broth  in  a  test-tube.  The  mixture  is 
inoculated  with  a  drop  of  a  typical  Bacillus  typhosus  culture. 
The  tube  is  then  incubated  at  37°  C.  for  twenty-four  hours,  after 
which  it  is  examined.  If  the  reaction  be  positive,  the  broth  ap- 
pears comparatively  clear,  but  at  the  bottom  of  the  test-tube  a 
more  or  less  abundant  sediment  will  be  found.  This  is  due  to 
the  clumps  of  bacilli  having  fallen  owing  to  gravity.  If,  on  the 
other  hand,  the  reaction  is  negative,  the  broth  will  appear  more 
or  less  uniformly  turbid. 

For  the  apparatus  required  to  carry  out  the  simpler  methods  of 
bacteriological  work  reference  should  be  made  to  the  standard 
laboratory  text-books,  which  furnish  all  necessary  details.  A 
good  microscope,  with  a  ^  oil  immersion  lens,  is,  of  course,  essen- 
tial. This  can  now  be  obtained  for  about  £\ 6  (Beck,  Swift,  Baker, 
Watson,  etc.),  and  the  other  necessary  apparatus  is  readily  obtain- 
able of  Baird  andTatlock,  Hatton Garden,  E.G.,  and  other  makers. 


INDEX 


Abscess  formation,  296-301 

Acetous  fermentation,  115,  127 

Actinomycosis,  316 

Aerobic  organisms,  26 

Agar,  21 

Air,  bacteriology  of,  96-110 

—  examination  of,  96-99 

—  of  sewers,  105 

—  expired,  102 

—  bacteria  and  gravity,  106 

—  standard  of  bacteria  in,  108 

—  pathogenic  bacteria  in,  109 

—  passages,  bacteria  in,  103 
Alcohol,  formation  of,  115 
Alcoholic  fermentation,  115,  117 
Alexines,  249,  268 

Alformant  lamp  for  disinfection,  334 
Algae  in  water,  53 
Ammoniacal  fermentation,  115 
Amylolytic  ferments,  115 
Anaerobic  organisms,  132 

—  methods  of  culture,  139-142 

—  in  hydrogen,  139,  140 

—  in  glucose-agar,  142 

—  in  Frankel's  tube,  140 

—  in  Buchner's  tube,  141 
Aniline  dyes,  44 
Antagonism  of  organisms,  33 
Anthrax,  19,  26,  30,  34,  245 

—  pathology  of,  301 

—  spores  of,  302 

—  bacillus  of,  302 
Antiseptics,  323,  332 

—  definition  of,  322 

—  some  of  the  chief,  332 
Antitoxins,  245-250 

—  preparation  of,  259 

—  use  of,  263 

—  unit  of,  263 
Appendix,  337 
Arthrospores,  17 

Artificial  purification  of  water,  73 


Ascospores,  120 
Asiatic  cholera,  65 
Association  of  organisms,  31 
Attenuation  of  virulence,  36 

Bacillus,  definition  of,  n 

—  aceti,  34,  129 

—  acidi  lactici,  131,  185,  190 

—  amylobacter,  132 

—  anthracis,  26,  31,  34,  no,  301-305 

—  aquatilis,  53 

—  butyricus,  133 

—  coli  communis,  32,  56,    58-62,  64, 

67,  86,    88,    108,    151,    194,  237, 

299,  315 

—  cyanogenus,  193 

—  diphtheriae,  201,  212,  244,  289-296 

—  enteriditis  sporogenes,   60,  86,  87, 

3i6 

—  erythrosporus,  53 

—  fluorescens  liquefaciens,  43,  53,  64, 

86 

—  fluorescens    non-liquefaciens,     53, 

151 

—  of  cholera,  65-69 

—  of  diarrhoea,  203,  204 

—  of  influenza,  315 

—  lactis  erythrogenes,  193- 

—  lactis  pituitosi,  193 

—  lactis  viscosus,  193 

—  liquefaciens,  53,  151 

—  of  leprosy,  308-313 

—  of  glanders  (mallei),  299,  319 

—  mesentericus,  86,  151 

—  mycoides,  151 

—  of  malignant  oedema,  19,  172 

—  No.  41,  219 

—  pasteurianum,  130 

—  of  scarlet  fever,  202 

—  of   symptomatic  anthrax,  19,  171, 

172 

—  of  plague,  306-308 


343 


344 


INDEX 


Bacillus    prodigiosus.    34,    151,    193, 
238 

—  pyocyaneus,  7,  34,  64,  no,  299 

—  pyogenes  fcetidus,  34 

—  radicicola,  164 

—  saponacei,  193 

—  subtilis,  31,  86,   108 

—  synxanthus,  194 

—  of  tetanus,  19,  168-171 

—  termo,  53 

—  of   tubercle,    no,   212,    228,   274- 

291 

—  typhosus,  41,  50,  55-62,  212 

—  ubiquitous,  53 

—  of  yellow  fever,  316 
Bacteria,  action  of,  26 

—  in  sewage,  84 

—  and  wheat  supply,  161 

—  and  fixation  of  nitrogen,  160-166 

—  in  cheese-making,  220-227 

—  in  the  dairy,  215-227 

—  products  of,  240,  241 

—  and  disease,  264-321 

—  the  higher,  n,  33 

—  in  soil,  137-177 
Bacterial  action,  26 

—  treatment  of  sewage,  90 
Bacteroids,  166 

Beer  diseases,  134 

Berkefeld  filter,  52 

Biogenesis,  3 

Biology  of  bacteria,  1-36 

Bitter  fermentation,  191 

Blood  serum,  22 

Blue  milk,  193 

Boracic  acid,  205,  331 

Bread,  bacteria  in,  238 

Broth,  21 

Brownian  movement,  14 

Bubonic  plague,  306-308 

Buchner's  tube,  141 

Butter,  bacteria  in,  213,  214 

—  examination  of,  214 

—  bacterial  flavouring  of,  215 
Butyric  fermentation,  115,  132,  191 

Carbol-fuchsin,  44 

Carbol-gelatine,  62 

Carbolic  acid  as  a  germicide.  332 

Caries,  dental,  104 

Chamber,  moist,  41 

Channels  of  infection  in  disease,  269 

—  in  tubercle,  289 
Cheese,  bacteria  in,  220 
Chemical  products  of  bacteria,  241 


Chemical  substances  as  disinfectants, 

329 

—  and  bacteriological  examination  of 

water  compared,  51 

—  tests  for  nitrification,  158 
Chemiotaxis,  15,  248 
Chicken  cholera,  320 
Chinosol  as  a  disinfectant,  336 
Chloride  of  lime  as  a  germicide,  331 
Cholera,  65-6$ 

—  diagnosis  of,  68 

—  and  filtration,  75 

—  and  milk,  200 
Chromogenic  bacteria,   193,  241 
Cladothrix,  8,  33 

Clark's  process,  73 
Classification,  7 
Coccus,  definition  of,  8 
Colon  bacillus,  see  B.  coli  communis 
Comma  bacillus,  66 
Commensalism,  162 
Composition  of  bacteria,  12-14 
Conditions  affecting  bacteria  in  water, 
70 

—  in  milk,  186,  187 
Contagion,  270 

Corrosive    sublimate  as   disinfectant, 

331 

Counter  (Wolfhugel),  49 
Cover-glass  preparations,  44 
Cream,  bacteria  in,  213 
Crenothrix  polyspora,  53 
Creosol  as  a  germicide,  332 
Cultivation  beds,  92 
Culture  media,  20 

—  anaerobic,  139 

—  hanging  drop,  44 

—  plate,  40-43 

—  pure,  20,  46 

—  shake,  62 

Decomposition  bacteria,  149 
Denitrifying  bacteria,  143,  149 
Dental  caries,  104 
Deodorants,  323 
Desiccation,  26 
Diagnosis,  339 

Diarrhoea  of  infants,  175,  316 
Diphtheria,  243-245,289-296 

—  bacillus  of,  289 

—  toxins  of,  244,  293 

—  and  milk  supply,  201 

—  and  school  influence,  294,  295 

—  pseudo-bacillus  of,  296 
Diplococcus,  definition  of,  8 


INDEX 


345 


Diplococcus  of  gonorrhoea,  300 
• —  in  pneumonia,  313 
Directions    for    estimating    disinfect- 
ants, etc.,  324 
Disease,  production  of,  264 
Diseases  of  beer,  134 

—  of  plants,  35 

—  of  animals,  316-320 

—  conveyed  by  water,  81 

—  and  soil,  173-177 
Disinfectants,  322,  331 
Disinfection,  322-336 

—  of  a  room,  335 

—  of  walls,  335 

—  of  bedding,  335 

—  of  garments,  335 

—  of  excreta,  335 

—  of  wounds,  336 

—  of  hands,  336 

Domestic  purification  of  water,  79 
Dunham's  solution,  69 
Dysentery,  319 

Earth  temperatures  and  disease,  175 

Economic  bacteria,  145 

Egg  cultures,  340 

Eisner's  medium,  62 

Endospores,  17 

Enteric  fever,  see  Typhoid 

Enzymes,  114 

Equifex  disinfector,  328 

—  sprayer,  334 
Erysipelas,  299 
Examination,  bacteriological — 

—  air,  96-99 

—  cholera,  68 

—  diphtheria,  290,  340 

—  leprosy,  309 

—  meat,  234 

—  milk,  227 

—  sewage,  80 

—  soil,  138 

—  tetanus,  170 

—  tubercle,  276,  339 

—  water,  43-48 

—  yeasts,  119,  et  seq. 
Extracellular  poisons,  244,  273 

Fermentation,  111-136 

—  acetous,  115,  127 

—  alcoholic,  115,  117 

—  ammoniacal,  115,  see  under  Soil 

—  butyric,  115,  132,  191 

—  lactic  acid,  115,  130,  190 
Ferments,  organised,  114,  115 


Ferments,  unorganised,  114,  115 

—  chromogenic,  193 

—  curdling,  191 

—  bitter,  191 

—  slimy,  192 

—  soapy,  193 
Films,  123 
Filter,  domestic,  79 
Filter-beds,  74 
Filtration,  milk,  206 

—  method  of  air  examination,  99 

—  sand,  77-79 
Fission,  16 

Fixing  specimens,  45 
Flagella,  15 

—  staining,  63 

Food,  bacteria  in,  179,  180 
Foot-and-mouth  disease,  320 
Formaldehyde  and  formalin,  205.  206, 

333 

Forms  of  bacteria,  8 
Frankel's  tube,  140 

—  pneumococcus,  314 
Friedlander's  pneumo-bacillus,  315 

Gas,  production  of,  62,  241 
Gathering-ground,  38 
Gelatine,  21 

—  carbol,  62 

—  liquefaction  of,  44 
Gemmation,  119 
Gentian-violet,  aniline,  44 
Germicidal  temperatures,  30,  207 
Germicides,  322,  331 
Glanders,  319 

Gonorrhoea,  300 
Gram's  method,  44,  338 
Gravity,  influence  on  bacteria,  106 
Gypsum  block,  121 

Hsematozoa,  320 

Hanging  drop  cultivations,  44 

Hansen's  method  of  dilution,  123 

Heat  as  steriliser,  30,  326 

Heredity,  268,  269 

Hesse's  method  of   air  examination, 

98 

Higher  bacteria,  n 
High  yeasts,  125 
Hot  air  steriliser,  31 
Hydrogen  cultivation,  139 
Hydrophobia,  treatment  of,  253 

Ice-cream,  bacteria  in,  236-238 

—  examination  of,  236 


346 


INDEX 


Immunity,  240-263 

—  acquired,  247,  250 

—  active,  250 

—  artificial,  250 

—  natural,  250 

—  passive,  250 
Incubation  period,  271 
Incubators,  22 

Indol,  formation  of,  61 

—  testing  for,  61 
Industries  and  bacteria,  135 
Influenza,  315 
Intracellular  poisons,  34 
Inversive  ferments,  115 
Involution  forms,  12,  66 

Kipp's  apparatus  for  producing  hydro- 
gen, 27,  139 

Klebs-Loffler  bacillus,  289 
Koch's  plate  method,  40 

—  postulates,  266 

—  comma  bacillus,  66 

—  bacillus  of  tubercle,  276 

Lactic   acid  fermentation,    115,    130, 

185,  190,  221,  226 
Lactose,  190 
Leguminosae,  fixation  of  nitrogen  by, 

163 

Leprosy,  308-313 
Leptothrix,  8,  33 
Leuconostoc,  17 
Light,  influence  upon  bacteria,  24-26, 

70 

Liquefaction  of  gelatine,  44,  241 
Low  yeasts,  125 
Lymph,  glycerinated  calf,  252 
Lyon's,  Washington,  disinfector,  328 

Maceration  industries,  135 
Malaria,  177,  320 
Malignant  oedema,  19,  172 
Mallein,  319 
Mastitis,  184 
Measles,  321 
Meat,  234 
Media,  culture,  20 
Merismopedia,  n 
Method  of  examination,  43-47 
Metropolitan  water  supply,  72 
Miasmatic  diseases,  176 
Micrococcus,  definition  of,  8 

—  agilis,  16 

—  aquatilis,  53 

—  casei  amari,  226 


Micrococcus  Freudenreichii,  192 

—  gonorrhoea,  299  • 

—  tetragonus,  299 

—  viscosus,  192 

Milk,  bacteriology  of,  178-213 

—  absorptivity  of,  180 

—  sources  of  pollution,  181-184 

—  number  of  bacteria  in,  185 

—  influence  of  temperature  upon,  186 

—  influence  of  time  of  standing,  187 

—  fermentation  bacteria  in,  189 

—  constitution  of,  189-195 

—  disease-producing  power  of,  195 

—  and  tuberculosis,  197-199,  228,  290 

—  and  typhoid,  199 

—  and  cholera,  200 

—  and  diphtheria,  201 

—  and  scarlet  fever,  202 

—  and  thrush,  203 

—  methods  of  preservation  of,  205 

—  and  added  antiseptics,  205,  206 

—  filtration  of,  206 

—  sterilisation  of,  207 

—  pasteurisation  of,  208-213 

—  products,  bacteria  in,  213-227 
— •  examination  of,  227 

—  and  economic  bacteria,  215-220 

—  sterile,  181 

—  kinds  of  bacteria  in,  188 

—  chromogenic,  193 

—  cooling  processes,  207,  209 
Moist  chamber,  41 

Moisture  necessary  for  bacteria,  23 
Motility,  14 
Moulds,  116 
Mycoderma  aceti,  127 

Nasal  passages,  bacteria  in,  103 
Natural  purification  of  water,  69 
Needles,  platinum,  22,  24 
Nitrates,  147,  154 
Nitric  organism,  157,  158 
Nitrification,  76,  143-159 

—  chemistry  of,  144-148 

—  stages  in,  145 

—  bacteria  of,  152 

Nitrifying  organisms,   cultivation  of, 

156 

Nitrogen,  fixation  of,  144,  160-168 
Nitrous  organism,  154,  158 
Nodules  on  roots,  bacteria  in,  163 

Oidium  albicans,  203 

Oxygen  necessary  for  bacteria,  26 

Oysters  and  bacteria,  229-234 


INDEX 


347 


Paraform  for  disinfection,  334 
Parasitism,  27,  162 
Parietti's  method,  62 
Pasteurisation  of  milk,  208-213 
Pasteur's  treatment  of  rabies,  253-258 
Perlsucht,  283 
Petri  dishes,  50 
Phagocytosis,  247 
Phosphorescence,  26,  241 
Pigment,  formation  of,  241 
Place  of  bacteria  in  nature,  5 
Plague,  306-308 
Plant  diseases,  35 
Plate  cultures,  40-46 
Platinum  needles,  22,  24 
Pleomorphism,  12 
Pneumo-bacillus,  315 
Pneumococcus,  313 
Pneumonia,  313 
Polymorphism,  12 
Postulates,  Koch's,  266 
Potato  medium,  22 
Pouchet's  aeroscope,  96 
Proteolytic  ferments,  115 
Proteus  family,  86,  180,  297 

—  vulgaris,  60,  86,  151,  194 

—  zenkeri,  60 

Pseudo-diphtheria  bacillus,  296 
Ptomaines,  179 

Pure  culture,  20,  46 
Purification  of  water — 

—  natural,  69 

—  artificial,  73 
Pus,  296 

Putrefaction,  143-149 
Pyocyanin,  299 
Pyoxanthose,  299 

Quantitative  standard  for  water  bac- 
teria, 48,  49 

—  air  bacteria,  107 

—  milk  bacteria,  188 

—  soil  bacteria,  137 
Quarter  evil,  19,  171 

Rabies,  treatment  of,  253 

—  forms  of,  253 

—  pathology  of,  254 

—  results  of  treatment,  256 
Reek's  disinfector,  328 
Reproduction    of    bacteria,    methods 

of,  16 

Retting,  135 
Rinderpest,  320 


Sacchardmycetes,  biology  of,  119-121 

—  methods  of  examination,  122 

—  anomalous,  122 

—  apiculatus,  127 

—  aquifolii,  127 

—  cerevisise,  117,  124,  126 

—  conglomerate,  126 

—  ellipsoideus  I.,  126 

—  ellipsoideus  II.,  126,  134 

—  exiguus,  127 

—  Hansenii,  127 

—  illicis,  127 

—  Ludwigii,  122 

—  mycoderma,  127 

—  pastorianus  I.,  127,  134 

—  pastorianus  II.,  127 

—  pastorianus  III.,  127,  134 

—  pyriformis,  127 

Salicylic  acid  as  antiseptic,  205,  206 

Saprophytes,  27,  166 

Sarcina,  10 

Scarlet  fever,  202,  321 

Sedgwick's  method  of  air  analysis,  99 

Sedimentation,  71,  73 

Septic  processes,  296 

—  tank,  90 

Sewage,  organisms  in,  84 

—  bacterial  treatment  of,  89 
Sewer  air,  87 

—  and  toxicity  of  bacteria,  105 
Shake  culture,  62 

Shell-fish  and  bacteria,  229,  234 
Small-pox,  251,  321 
Soil,  bacteriology  of,  137 

—  examination  of,  138 

—  kinds  of  bacteria  in,  142-145 

—  and  its  relation  to  disease,  173,  176, 

177 

Species  of  bacteria,  29 
Spirillum,  definition  of,  n 

—  of  cholera,  66 
Spontaneous  generation,  2 
Spores,  kinds  of,  17-19 

—  resistance  of,  19,  278 

—  staining  of,  19 

—  of  yeasts,  122 
Staining  methods,  45 
Staphylococcus,  10,  296 

—  cereus  albus,  297 

—  pyogenes  aureus,  88,  108,  297 
Steam  as  a  disinfector,  326 

—  disinfectors,  327-329 

—  steriliser,  326 

—  saturated,  327 

—  superheated,  327 


348 


INDEX 


Steam  current,  327 
Sterilisation,  29-31 

—  methods  of,  30,  31 
Streptococcus,  g,  297 

—  pyogenes,  299 

—  Hollandicus,  192 
Streptothrix,  317 
Structure  of  bacteria,  8 
Sulphurous  acid  as  a  germicide,  332 
Suppuration,  296-301 

Swine  fever,  320 
Symbiosis,  162 
Symptomatic  anthrax,  171 

Table  of   economic  bacteria  in   soil, 

145 

Temperature,   influence  of,    on   bac- 
teria, 23 

Tetanus,  19,  168,  245 

—  toxin  of,  168 
Thresh's  disinfector,  328 
Thrush,  203 

Tissues,  effect  of,  on  bacteria,  267 
Tobacco-curing,  136 
Toxins,  28,  241-247,  272 
Tuberculin,  281 
Tuberculosis,  274-292 

—  pathology  of,  274 

—  varieties  of,  275 

—  history  of,  276 

—  conveyed  by  the  air,  104 

—  and  the  milk  supply,  196-198,  290 

—  giant  cells  in,  275 

—  bacillus  of,  276 

—  cultivation  of  bacillus  of,  277 

—  spores  of,  277 

—  relation  of  bacillus  to  disease,  279 

—  toxins  of,  281 

—  of  animals,  283 

—  prevention  of,  286-292 

—  disinfection  in  cases  of,  292 

—  decline  of,  288 

—  and  overcrowding,  288 

—  channels  of  infection  in,  289 

—  expectoration  in,  290 


Tuberculosis  and  house  influence,  291 

—  and  glanders,  319 
Typhoid  fever,  56 

—  bacillus  of,  55-58 

—  micro-pathology,  57 

—  bacillus  compared  with  B.  coli,  58 

—  bacillus  in  sewage,  59 

—  bacillus  in  drinking  water,  60 

—  tests  for  bacillus  of,  61-64 

—  and  soil,  173,  176 

—  conveyed  by  the  air,  105 

—  and  milk  supply,  199 
Tyrotoxicon,  205,  237 

Unit  of  antitoxin,  263 
Urea,  148 

Vaccination,  251-253 
Vaccines,  plague,  257,  259 

—  cholera,  257 

—  small-pox,  251 
Vaccinia,  251 
Variolation,  250,  251 
Virulence  increased,  32 

—  diminished,  36 

Water,  bacteria  in,  37-84 

—  number  of  bacteria  in,  38 

—  examination  of,  39-52 

—  disease  organisms  found  in,  55 

—  natural  purification  of,  69 

—  artificial  purification  of,  73 

—  filtration  of,  74 

—  domestic  purification  of,  79 

—  pollution  of,  82 

Wheat  supply  and  bacteria,  161 
Widal  reaction,  63,  340 
Wooden  tongue,  318 
Wool-sorters'  disease,  305 

Yeasts,  116 
Yellow  fever,  316 

Ziehl-Neelsen  stain,  44,  340 


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